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Factors Affecting Fertility with Artificial Insemination
FEMALE BOVINE INFERTILITY
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FACTORS AFFECTING FERTILITY WITH ARTIFICIAL INSEMINATION Albert D. Barth, DVM, M Vet Sci
The reproductive performance of individual beef or dairy herds is extremely important and plays a major role in the financial success of livestock enterprises. Economically, the most important reproductive goal of beef and dairy herds is that each cow must calve on a yearly basis. Most beef cows are managed on a seasonal basis and must calve every 12 months in a defined calving season. Failing to do so leads to a nonpregnant status or an expected late calving date and such cows must be culled to maintain herd reproductive efficiency. Most commercial beef cattle and a large proportion of purebred beef cattle are bred naturally, and the use of highly fertile bulls is of utmost importance. A significant number of beef cattle, particularly in purebred herds, however, are bred by artificial insemination (AI). Cows often are given one chance to become pregnant via AI and then are exposed to "clean up" bulls for the remainder of the breeding season. Success with AI in such herds is highly dependent on accurate heat detection, use of highquality semen, and proper insemination technique. In North America, most dairy cows are not managed to freshen on a seasonal basis, but to maintain a high level of productivity, dairy cows also must calve yearly. Most dairy herds would find a goal of a 12-month calving interval difficult to achieve, however, and the economics of such a goal may be questioned as well. Some high producing dairy cows may be most productive with a somewhat longer calving interval; a 12.5- to 13-month herd calving interval may be a more realistic goal. Unlike beef cattle, most dairy cows are bred by AI. Low From the Department of Herd Medicine and Theriogenology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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breeding efficiency is one of the most serious economic problems confronting dairy management. 26, 27 When conception is delayed and the calving interval is lengthened, milk revenue usually is reduced because cows spend a greater portion of their lactation at low production levels. Dairy and beef cows that fail to conceive or that conceive too late to justify their retention in the herd must be sold for slaughter, often at a much lower value than their purchase price or rearing cost. The costs of AI increase in proportion to the additional breedings required, and the cost of veterinary services and medication in repeat breeding cows also may be increased. Additional losses due to delayed genetic progress can be substantial, although impossible to calculate. Breeding problems tend to discourage the use of bulls with a high predicted difference that demand a premium price for semen, particularly for repeat services. Cows with low productivity must be culled to maintain economic efficiency, but this culling potential is greatly reduced in herds with breeding problems because of the number of cows that must be sold due to breeding failure and because fewer replacements are born. In general, any herd consists of three kinds of cows, classified according to reproductive status-those that are pregnant, those that have calved within the past 45 days (the period for uterine involution and resumption of estrous cyclicity), and those that must be rebred for the next gestation. Every cow in the herd falls into one of these three categories. Cows in the first two categories cannot contribute to the herd's current reproductive problems. If a yearly calving interval is desired, then cows with a 285-day gestation period and a 45-day postpartum period have 35 to 50 days in which to become pregnant. It is obvious that an excessive number of days in the breeding period is incompatible with a high reproductive rate. The average number of days open for all cows in the herd therefore is a good measure of the current reproductive status of the herd. There are many reasons for failure of cows to become pregnant and an excessive accumulation of days open." The two most important are cows that are not bred and cows that must be rebred. Organic conditions such as uterine infections, cystic ovaries, and abortions generally make only minor contributions to days open. More commonly, reproductive problems in a herd are attributable to management problems. Lack of ovarian cyclicity or low conception rate in cycling dairy cows because of high milk production and a negative energy balance are important herd management problems, but as many as 90% of anestrus cases are due to failure in heat detection. 47 With good management, AI can be highly successful if semen is placed into the cow's uterus near the time of ovulation. In this regard, efficient, accurate heat detection is of paramount importance. In addition, because conception rates are low when inseminations are done improperly, proper handling of semen and proper insemination technique are equally important. JI
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THE STATUS OF ARTIFICIAL INSEMINATION IN NORTH AMERICA
Artificial insemination offers several distinct advantages over use of natural mating, and this has resulted in the development of a highly successful AI industry. The use of AI on a commercial basis developed quickly after World War II. Most semen initially was chilled liquid semen with a short life span. One of the major advantages of AI is to extend the use of superior sires over a higher number of females than would be possible with natural service. With the advent of storage of frozen semen in the 1950s, sire testing programs became more feasible, allowing for more intensive genetic selection and rapid genetic improvement. Other potential advantages of AI include the elimination of bulls with fertility problems and bulls with unexpected variations in fertility. Strictly controlled health regulations of AI bulls have prevented disease transmission between females, which occurs more readily when natural service is used. Artificial insemination also has the advantage of improved safety on farms through the elimination of bulls, particularly dairy bulls, which frequently caused injury and death. The percentage of cattle bred by AI in North America varies among regions but includes approximately 75% of dairy cows and 5% of beef cows on a national basis for both the United States and Canada (Canadian Association of Animal Breeders, and National Association of Animal Breeders, personal communication, 1992). The use of AI in the beef industry has developed slowly and is still limited in commercial herds, but it now is used fairly extensively in purebred herds. In the past 10 to 15 years, there has been a significant increase in the number of breeders storing frozen semen on the farm and breeding their own cows. A very substantial portion of the cows are still inseminated by professionals employed by AI organizations, however. These organizations also provide training courses in insemination and provide semen sales and delivery to the farm. The AI industry has worked diligently to improve cryopreservation technology and AI centers strive to provide a high quality product that will perform well if used correctly. When healthy fertile cows are inseminated with frozen semen from highly fertile bulls at the correct time in their estrous cycle and the semen is properly placed into the uterine body, pregnancy rates similar to those seen with natural service can be expected. In three different studies2, 8, 14 in which heifers were slaughtered 3 to 5 days after AI, the fertilization rate was 90% to 100%. However, AI is a technical procedure under human influence and many factors may adversely affect pregnancy rates obtained by AI. Calving rates are substantially lower than fertilization rates because cows are not always in excellent health or mated at the proper time and not all conceptuses become established in the uterus or grow normally to parturition. In general, AI centers today average 60- to 90-day nonreturn rates of about 70%. The nonreturn rates, when calculated for large numbers of inseminations, are reliable measures for comparison of relative fertility among individual bulls or inseminators, but they
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substantially overestimate the true calving rates. In a Minnesota study, conception rates for 2270 services in 17 dairy herds during 1980 and 2217 services in 18 dairy herds in 1982 and 1983 were 41% and 39% respectively.40 Studies in New York and California have both shown that the true calving rates to first services were around 50%, even though the AI centers routinely reported 60- to 90-day nonreturn rates of 65% to 75%. Pellissier27 reported that the conception rate from 12,964 first services in California dairy herds was 44.2%. In the same study, only 57.7% of the cows conceived in the usually accepted optimum 61to 120-day postpartum period. In a New York survey, data from 9750 cows in 125 dairy herds on Dairy Herd Improvement records showed a 50% pregnancy rate after one service compared with a 58% nonreturn rate. 44 The yearly average 60- to 90-day nonreturn rate in herds using the same AI service was 69%. The two principal factors identified in this study accounting for the difference between actual conception and 60- to 90-day nonreturn rate were cows that died or were disposed after a first service and never received a second service and cows that were presented for a second service beyond the 60- to 90-day postbreeding interval.
EFFECT OF TIME OF INSEMINATION ON PREGNANCY RATE ON FERTILITY
The general recommendation has been to breed cows in the middle to the end of standing .heat for optimum fertility. Because the period of estrus may vary from 6 to 24 hours, however, it is difficult to determine when the midpoint is reached. The general guideline for determining insemination time originated in a 1948 study by Trimberger,45 in the form of the AM-PM rule-i.e., if cows are first observed in heat in the morning (AM) they should be bred that afternoon (PM); if they are first seen in heat in the late afternoon (PM), they should be bred the next morning (AM). In the Trimberger study, the time of breeding was divided into nine different time periods with 16 cows per breeding period. Five time groups were used to separate the 24 hours prior to ovulation. The overall conception rate was 69.4% (50172) and the best time for breeding was 13 to 18 hours prior to ovulation (12/14 for an 85.7% pregnancy rate). The time relationships concerning estrus, luteinizing hormone (LH) release, and ovulation in 61 heifers and cows were reported by Schams et al in 1977. 39 In their study, the surge of LH peaked at 6.4 ± 3 hours after the onset of estrus and ovulation occurred at 25.7 ± 6.9 hours after the LH peak. Their data also indicated that, although the preovulatory LH peak occurred at any time during the day, it peaked predominantly at around 8 AM or 8 PM. As a result, there were two main periods for ovulation. According to these data, insemination at 12 hours after the onset of estrus would result in insemination at about 20 hours prior to ovulation. This agrees fairly well with the AM-PM rule, which would result in insemination at the
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optimum time suggested by the Trimberger study of approximately 13 to 18 hours prior to ovulation. Although one study 17 found that conception rates in 367 heifers were higher when breeding occurred in 2 to 6 hours after onset of estrus than when inseminations took place 10 or more hours after the onset of estrus, other recent studies have supported the AM-PM rule. MacMillan and Watson,21 in New Zealand, examined over 6000 inseminations to determine the effects of stage of estrus at the time of breeding on conception rates. Inseminations were done in the morning with unfrozen extended semen collected the previous day using 2.5 million total cells per dose. The mean pregnancy rates for all sires, as determined by 18- to 49-day nonreturn rates, were 65.7%, 69.3%, 75.1%, and 72.8%, respectively for breedings made in early, mid, and late estrus or postestrus. For sires of above average fertility, nonreturn rates for the different insemination times tended to be higher for late estrus inseminations but were not statistically different. For bulls of average or low fertility, nonreturn rates for the four insemination times differed significantly. In another study, 12 in which dairy cows were inseminated either immediately after observed estrus (358 inseminations) or 12 hours after observed estrus (360 inseminations), the pregnancy rates were 51.1% and 55% for O-hour and 12hour breedings, respectively, but the difference in pregnancy rate was not statistically significant (P> .05). In a study involving 2091 first inseminations in beef cows on range, pregnancy rates were compared for successive 2-hour periods from estrus detection to insemination. The overall first insemination calving rate was 63% for cows inseminated between 12 and 29 hours after estrus detection. The highest pregnancy rates occurred for inseminations done 16 to 25 hours after estrus detection. 33 From these studies it would appear that the best time for insemination in the cow is 12 to 20 hours after the first observation (onset) of estrus. When heat detection is done in the morning and evening, therefore, the AM-PM rule established by Trimberger would be appropriate.
THE EFFECT OF VARIATION IN SEMEN QUALITY ON FERTILITY
Considerable research information has been generated over the past 40 years on all aspects of cryopreservation technology and semen quality requirements for successful AI in cattle. Although industry standards for semen quality are not uniform and are not regulated, the commercial viability of AI centers depends on the production of a highquality product that performs well in the hands of the consumer. Artificial Insemination centers have been leaders in research and development and have built a highly reputable industry. Nevertheless, AI centers, particularly those that derive a great deal of their revenues from custom collection of beef bulls, often are under a great deal of pressure to preserve semen of marginal quality. In many cases, cattle
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breeders have invested a great deal of money in a bull and must sell semen as quickly as possible to recover their investment. Many young beef bulls are still undergoing puberty and are still producing lowquality semen when cryopreservation is begun. These young beef bulls, as well as older beef bulls entering AI centers, often have been overfed and have been under considerable stress associated with the show circuit. They often require several months to adjust to the new environment of an AI center and need to lose body weight and fat from the scrotum before they are capable of producing good-quality semen. However, cattle breeders often are adamant that their bulls must maintain or gain weight during semen collection because high body weight generally is seen as a reflection of the growthiness of a bull-a desirable trait that it is hoped he would pass on to his offspring. Beef bulls frequently are selected for growth rate, height, coat color patterns, and other physical characteristics not related to fertility. Although these bulls may be show winners and bring the highest prices at prestigious sales, they may have abnormalities such as testicular hypoplasia, epididymal anomalies, and hereditary sperm defects that preclude them from ever being able to produce good semen. Furthermore, many beef cattle breeders are willing to accept a reduced level of semen performance in order to obtain the desired genetics from a popular bull. Thus, there is a ready market for a certain amount of semen that is of marginal quality. Most dairy bulls are owned by AI centers and not subject to many of the reproductively detrimental influences experienced by beef bulls destined for AI. In general, semen from dairy bulls is less likely to be of marginal quality than that of beef bulls. Those involved in the embryo transfer industry must be aware that semen that results in lower but perhaps acceptable fertility in single ovulating females usually performs more poorly when used in superovulated donor cows. Because of the high costs involved in embryo collection and transfer, only semen of high fertility should be used, and it would seem prudent to evaluate each freeze batch of semen prior to superovulation of the donor cow. The validity of laboratory assays for prediction of fertility of frozen semen often is questioned, and correlations of specific semen traits with fertility have ranged from very low to very high. 5- 7, 9, 19, 46 One therefore might conclude that little is known about semen quality as it relates to fertility. However, the inability to obtain high correlations between semen quality traits and fertility does not necessarily mean that semen evaluations lack validity. Indeed, many experiments that were unable to demonstrate a correlation between semen quality and fertility lacked validity themselves. In general, the reasons for low correlations between semen quality traits and fertility rest largely in the inability of experiments to account for experimental error. Some examples include: 1. The assessment of the effect of single traits of semen on fertility does not take into account variability caused by other traits. The effects of differences in percentage of progressively motile spermatozoa on fertility, for example, may be masked by unaccounted for variation in
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sperm morphology or sperm dose. When these sources of error are not accounted for, differences in fertility due to a trait such as sperm motility are minimized and resulting correlations are low. 2. There may be insufficient difference between the quality traits under assessment to result in a difference in fertility. As the quality of semen increases, the resultant fertility increases until the highest fertility potential of the female population is reached. Further increases in semen quality would not result in any further increase in fertility. 38 When all differing levels of semen quality traits being assessed are adequate for optimum fertility in the female population, therefore, a low correlation for the trait measured results. Indeed, in many of the reported fertility trials, the semen that was compared with control semen had not been rejected because it had met the minimum quality requirements of the AI center. Linford et aP9 demonstrated the importance of using semen with large differences in quality for demonstrating correlations between laboratory tests and fertility. When bulls with relatively low variation in fertility were used, correlations between semen quality tests and fertility were low. When a second group of bulls, in which there was a larger variance in semen quality, was used, correlations between semen quality tests and fertility were greatly improved. 3. The most commonly used measure of fertility in semen quality research has been the nonreturn rate (NRR) of cows to estrus after insemination. Before the days of direct semen sales and owner-producer inseminators, each AI center had a quick and inexpensive way of retrieving information on semen performance in the field. The NRR, however, has some inherent sources of error that are difficult to control. Recordkeeping and reporting often are not strong points in the management of herds. Many producers use a herd bull for some of the breeding and bulls have always had the notoriety of unexpectedly producing calves from cows thought to be pregnant via AI. Cows that do not conceive after one or more inseminations may be culled but would appear in the NRR data not to have returned to heat. Similarly, cows that return to heat a second or third time may be rebred with semen from different AI centers. Each center would receive receipts for only one breeding and assume a first-service conception. Heat detection efficiency often does not exceed 50%. Hoffmann et al13 reported that cows that show no behavioral estrus after insemination although they are not pregnant, and those inseminated during the luteal phase (and thus "not returning to estrus") comprise between 20% and 27% of cows inseminated. Most NRR data are obtained for the 60- to 90-day period after insemination(s). The difference between conception rate and NRR increases as the number of inseminations per cow increases. This difference was reported to be 15.9% for cows receiving a third service. 24 Therefore, although the NRR system of estimating fertility allows ranking of the relative fertility of bulls, it is inherently inaccurate as a measure of conception rate. Furthermore, it has a tendency to enhance the apparent fertility of low fertility semen, thereby reducing the apparent variance between good and poor semen. As a result, the
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correlation between semen quality traits and fertility measured by the NRR appears lower than it actually may be. Saake36 used heterospermic insemination (competitive fertilization) to investigate the validity of laboratory tests of semen quality to predict the fertility of frozen semen. Heterospermic insemination refers to the use of sperm from more than one male in an insemination dose. Semen usually is mixed such that sperm numbers from each competing male are equal and it is presumed that paternity of offspring should also be equal unless there is a difference in semen quality among competing males. The ratio of offspring based on paternity is converted to a competitive index. It has been shown that males that are highly fertile homospermically (high fertility when used under normal circumstances) also are superior heterospermically. 3 Large differences among males detected heterospermically, however, frequently are very small or not apparent when these bulls are tested homospermically. In addition to the improved variance in fertilizing potential offered by heterospermic insemination, this technique also minimizes or eliminates the masking influences of variation in female fertility and other biologic or environmental factors that may prevail at the time of insemination. Saake et al,36 using frozen bovine semen, obtained relatively high correlations between eight different semen quality measurements and competitive indexes from heterospermic inseminations. It has long been recognized that the repeatability of semen quality tests is not high among laboratories and even within laboratories. 11 This is particularly true for subjective assays. The use of computer assisted semen analysis has allowed objective assessment of semen quality traits and should improve repeatability, but the cost of the equipment may be out of reach or unjustified economically for many enterprises. At the present, therefore, we must continue to rely on subjective assessment of semen quality.
THE EFFECT OF SEMEN HANDLING ON FERTILITY
One of the major causes of low fertility associated with AI is mishandling of semen. This fact has been confirmed by studies involving professional inseminators closely supervised by AI organizations. 29-31 It seems likely that this problem would be accentuated on the farm and ranch by herd-owner inseminators who often have minimal training and no supervision. Over time, even professional inseminators may develop bad habits and a relaxed attitude toward semen handling and insemination technique, resulting in declining fertility. Several studies indicate the detrimental effects of exposure of frozen semen to ambient temperatures. The key factor to long-term cryopreservation is the very low (-196°C) temperature of the liquid nitrogen (LN) tank. Many do not realize the extreme importance of keeping exposures of semen to ambient conditions to an absolute minimum. From available evidence,
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it appears that temperatures should be maintained at -130°C or lower at all times. 16, 23, 32 Above this critical temperature, a phenomenon called recrystallization may occur, resulting in damage to cellular structures. During the freezing process, pure water is removed from solution in the formation of ice crystals and solutions increase in concentration. The resulting changes in osmotic pressures remove most of the intracellular water from spermatozoa or embryos. This dehydration occurs early during the freezing process and is essential to survival of cells during cryopreservation. Because of the rapid rate of freezing of spermatozoa, the remaining intracellular water forms very small ice crystals that appear to be relatively harmless. At temperatures above - 130°C, however, molecules of WOlter may leave ice crystals and reattach to other crystals, resulting in an increase in the size of some crystals at the expense of others (recrystallization). The larger crystals are potentially damaging to cellular structures. 32 Another source of damage, which occurs at intermediate temperatures above - BO°C, is the highly concentrated solution (solution effect) that results during the freezing and thawing process. 20, 22, 23 Injury is believed to occur by salt solution denaturation of specific enzymes and cell membranes, or loss of structural integrity when the cell shrinks below a minimum volume. 1 The relatively large surface area and small volume of straws used in cryopreservation are conducive to rapid heat exchange. Spermatozoa in 0.2S-mL straws are particularly vulnerable to rapid rises in temperatures upon exposure to ambient conditions. In this regard, semen that formerly was predominantly packaged in I-mL glass ampules had a much larger margin of safely than straws during handling in field situations. This was illustrated in the work of Berndtson et al, 4 which showed that 0.2S-mL and O.S-mL straws held by forceps and exposed to ambient air temperatures of 20 ± 0.6°C reached the temperature of recrystallization within 10 to 15 seconds. Under similar conditions, 1mL ampules clipped to metal canes required approximately 45 seconds to reach the temperature of recrystallization. Under field conditions, rates of warming upon exposure to ambient conditions may be much higher because of the effects of wind or sunshine. It is important to recognize that damage due to recrystallization is cumulative. Damage due to initial exposure may not be overt, but after repeated exposures, a reduction in post-thaw viability may become evident. Incubation of semen for 2 to 4 hours post-thaw may reveal such damage more clearly than immediate post-thaw examinations. Clearly, individual straws should never be exposed to ambient temperatures. Straws within goblets, particularly when the level of LN in the tank is high enough to fill the goblets, are much less susceptible to rapid temperature changes during removal of straws for AI or when transfers are made to other tanks. This was illustrated in the work of Berndtson et al, 4 which showed that when 10 0.2S-mL or 5 O.S-mL French straws per goblet were exposed to 20 ± 0.6°C ambient conditions, the temperature of recrystallization was not reached until 45 to 60 seconds of exposure.
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The danger of warming of semen occurs not only with exposure of straws to ambient conditions external to the LN tank. Exposure to potentially damaging temperatures occurs each time straws are raised to the neck of a tank to permit removal of an individual straw for thawing. In a report by Berndtson et al,4 the temperature within the neck of a LN semen storage tank was ranged from approximately - 180°C to + 2°C. Saake37 showed that the rate of warming to - 80°C was virtually identical for O.S-mL straws exposed to either - 22°C or + SoC temperatures. Therefore, canisters should be raised no higher within the neck of the LN tank than absolutely necessary and the individual straw should be removed as quickly as possible. In some instances, when large numbers of inseminations or embryo transfers are done sequentially, straws are repeatedly exposed to ambient temperatures. Unless sufficient time is allowed between exposures to allow cooling to - 196°C, higher temperatures will be reached during each exposure. When semen in straws placed in goblets on metal canes was raised to the top of the canister neck for five consecutive I-minute exposures, allowing I-minute returns to LN vapor between exposures, more than 10 minutes were required for straw temperatures to return to - 196°C after the fifth exposure. Ideally, the level of LN in the storage tank should be high enough so that the semen would be plunged back into the LN upon each return to the tank. 37 The foregoing studies suggest that semen stored and used in the field may be subject to gradual deterioration. In one study,25 test semen was placed in 39 LN field tanks and, over a period of 6 months, the semen was given at least 480 exposures to ambient conditions. When compared with those stored at the AI center, I-mL glass ampules showed no difference in viability of spermatozoa stored under field conditions. Significant deterioration in semen viability was found in semen packaged in O.S-mL straws, however. This difference was seen more readily after semen was incubated at 37°C for 2 to 4 hours. In addition, spermatozoa located in the top goblet had a significantly lower percent intact acrosomes than those in the bottom goblet. This study demonstrated that deterioration of semen in field units is quite possible. It is unlikely, however, that semen in field use would be exposed to the neck of the tank as frequently as in the foregoing study. In two subsequent studies,18, 41 test semen was placed in 60 semen tanks belonging to cooperating dairy producers. Semen was removed at 6-, 12-, 18-, and 24-month intervals and compared to semen of similar freeze batches that were stored at the AI center. In these studies, no difference was found in semen viability between on-farm and centrally stored semen. Furthermore, no difference in viability was seen between semen from top or bottom locations on the canes. This indicates that semen handled carefully in a routine farm situation should suffer no adverse effects. American Breeders Service in Colorado has developed a system to indicate whether temperatures within a tank have been raised to dangerous levels. Two ampules, each containing either blue or red liquid, frozen into the apex of the ampule are placed on a cane that is
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then placed with frozen semen in the LN tank. If temperatures rise sufficiently to thaw the colored fluid, it sinks to the bottom of the ampule. If only the blue ampule thaws, damage to the semen may not be very severe, but if both ampules thaw (blue and red fluid sinks to the bottom of the ampules), semen must be tested before use. The materials for this method of monitoring LN tank temperature are commercially available from Minitube of America Inc., Madison, Wisconsin. Sudden unexpected loss of LN from a tank containing embryos or semen may result in an economically disastrous loss. One report indicated that the failure rate of LN tanks in 57 dairy herds was approximately 5% over a 2-year period. 41 Liquid nitrogen tanks are expected to maintain a vacuum for at least 15 to 20 years if they are not abused, but it is not uncommon for new tanks to lose their vacuum even without any evidence of damage to the tank. There may be an indication that a tank is losing its vacuum when it seems to lose nitrogen at a faster rate than indicated by its specifications or when it needs to be replenished more frequently than usual; most tank failure is rather sudden, however. When vacuum is lost, all of the nitrogen may be lost from the tank within 24 hours. Rapid evaporation of nitrogen results in pronounced cooling of the top portion of the neck and this results in condensation of moisture from the atmosphere at the top of the tank. Depending on the rate of loss and whether the loss is detected while there is still LN in the tank, there will be a small build-up of frost or moisture at the top of the neck. Once all the nitrogen has evaporated, the neck warms up, the frost and moisture evaporate, and the tank looks like a normal tank. Usually, only the lid attached to the styrofoam neck piece becomes frosty or wet. The amount of frost or moisture may be very small, showing only light frosting on the parts of the lid touching the handles of the canisters below. New tanks and tanks that have been moved or shipped are more likely to suddenly lose vacuum than static holding tanks, but even static tanks have been known to lose vacuum suddenly. All tanks therefore must be monitored closely. Perhaps the only practical means of preventing loss of semen is to attach LN level sensors with warning indicators to every tank. Another means to ensure the preservation of at least half the semen is to divide it among two or more tanks, so if one tank fails, not everything is lost.
THE EFFECT OF INSEMINATOR EXPERTISE ON FERTILITY
In order to achieve optimum fertility with AI, inseminators must be able to place the semen into the uterus of the cow quickly and with a minimum of trauma to the cervix and endometrium. The junction of the cervix and the uterine body usually is recommended as the target for semen deposition in AI. This allows the semen to bypass the cervical
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barrier and should allow an equal number of sperm to enter each uterine horn. Thus, equal pregnancy rates are anticipated between the sides of the tract regardless which ovary contains the ovulatory follicle. Experiments have been done to determine whether better conception rates could be achieved by placing half the inseminate into each uterine horn (horn breeding) or by placing the inseminate on the side of ovulation rather than within the uterine body. In one study,40 the conception rate for 114 horn-bred cows was 67% versus 64% in 110 cows inseminated into the uterine body. In the same study, the conception rate in 44 cows and heifers inseminated on the side of ovulation was 70% compared with 75% for cows and heifers inseminated into the uterine body. The differences in fertility related to location of semen deposition were not significant, so, given that it is easier to deposit semen into the uterine body than up the uterine horn, the uterine body remains the best target site for semen deposition. Even so, previous work with dye testing showed that the AI target frequently is missed. 10, 15 The expertise of inseminators may vary according to the amount of training and practice as well as personal qualities that may influence the quality of work performed. In a study of 2820 first services, marked differences were reported in the performance of herdsmen inseminators.42 In this study, 10 proven AI bulls were used in four large commercial dairy herds. Other variables that might affect conception rates were controlled experimentally or accounted for statistically. Conception rate was determined by rectal palpation 35 to 45 days after insemination. The inseminator with the highest rate achieved a first-service conception rate of 63%, whereas the inseminator with the lowest rate achieved only 39%. Radiography has been used to visualize the interior of excised bovine reproductive tracts from packing plants to assess accuracy of semen placement. With this technique, reproductive tracts were placed in a device that simulated the anatomic position of the reproductive organs in vivo. Air was introduced to provide contrast between the uterine lumen and tissue and semen extender was treated with lead acetate to provide radiographic opacity of the inseminate. 28 This technique was used to evaluate and compare the insemination techniques of 20 professional and 20 herdsmen inseminators.43 Each inseminated 20 tracts. There was no difference in the ability of professionals and herdsmen inseminators to position the syringe tip in the uterine body. There was significant variation in correct syringe tip placement between individuals, however. Correct placement was achieved in 6% to 85% of cases by professionals and in 0 to 82% of cases by herdsmen inseminators. Fewer than half (39%) of placement attempts by all inseminators were in the uterine body. In 36% of placement attempts, the syringe tip was positioned in either the left or right uterine horn. The mean percentage of semen deposits within the cervix was 13% by professionals and 21 % by herdsmen inseminators. It would appear that marked improvements to AI fertility could be achieved through improved insemination technique. In recent times, with the use of minimal numbers of spermatozoa per insemination
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dose and smaller insemination volumes due to the introduction of 0.25mL straws, it is of utmost importance that semen be placed properly within the uterus. There are no routine testing or retraining programs for inseminators in the United States or Canada. In light of the results of studies such as the ones just mentioned,43 it appears that a great many herdsmen and professional technicians would benefit from routine retraining programs.
SUMMARY
Man's intervention in the natural processes of reproduction with the use of AI has allowed rapid genetic improvement in beef and dairy herds and has resulted in a marked increase in livestock productivity. In today's tough economic climate, high reproductive efficiency is of utmost importance for livestock enterprises to remain viable. Many factors affect the success of AI programs; of particular importance are the health and nutritional management of the herd and accurate, efficient heat detection. Good management combined with knowledge, technical expertise and careful attention to detail in the timing of insemination in relation to the period of estrus, semen handling, and correct semen placement in the uterus ensure the successful use of AI.
References 1. Arakawa T, Carpenter JF, Kita AK, et al: The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27:401-415, 1990 2. Bearden HJ, Hansel WM, Bratton RW: Fertilization and embryonic mortality rates of bulls with histories of either low or high fertility in artificial breeding. J Dairy Sci 39:312-318, 1956 3. Beatty RA, Bennett GH, Hall JG, et al: An experiment with heterospermic insemination in cattle. J Reprod Fertil 19:491, 1969 4. Bemdtson WE, Pickett BW, Rugg CD: Procedures for field handling of bovine semen in plastic straws. In Proc 6th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1976, pp 51-60 5. Bishop WH, Campbell RC, Hancock JL, et al: Semen characteristics and fertility in the bull. J Agric Sci 44:227-248, 1954 6. Bratton RW, Foote RH, Henderson CR, et al: The relative usefulness of combinations of laboratory tests for predicting the fertility of bovine semen. J Dairy Sci 34:1542, 1956 7. Buchner PJ, Willet EL, Bayley N: Laboratory tests singly and in combination for evaluating fertility of semen and of bulls. J Dairy Sci 37:1050, 1954 8. Diskin MG, Sreenen, JM: Fertilization and embryonic mortality rates in beef heifers after artificial insemination. J Reprod Fert 59:463-468, 1980 9. Elliott PI: Significance of semen quality. In Salisbury W, VanDemark NL, Lodge JR (eds): Physiology of Reproduction and Artificial Insemination of Cattle, ed 2. San Francisco, WH Freeman and Co, 1979, pp 428-441 10. Graham EF: The use of a dye indicator for testing, training and evaluating technicians in artificial insemination. In Proc 1st Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1966, pp 57-60 11. Graham EF, Schmehl MKL, Nelson DS: Problems with laboratory assays. In Proc 8th
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Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1980, pp 59-66 Gwasdauskass FC: The effect of time of insemination on conception rates in dairy herds. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, pp 92-96 Hoffmann B, Gunzler 0, Hamburger R, et al: Milk progesterone as a barometer for fertility control in cattle. Methodological approaches and present status of application in Germany. Br Vet J 132:469, 1976 Kidder HE, Black WG, Wiltbank IN, et al: Fertilization rates and embryonic death rates in cows bred to bulls of different levels of fertility. J Dairy Sci 37:691-697, 1954 King GJ, MacPherson JW: Observations on retraining artificial insemination technicians and its importance in maintaining efficiency. Can Vet J 6:83-85, 1965 Larson GL, Graham EF: Effects of low temperatures in storage of bovine semen. AI Digest 6:6, 1958 Laster DB, Glimp HA, Gregory KE: Age and weight at puberty and conception in different breeds and breed crosses of beef heifers. J Anim Sci 34:1031-1036, 1971 Lineweaver JA, Saake RG, Gwasdauskas FC: Effect of double decking and storage time on semen stored in farm storage tanks. In Proc Am Dairy Sci Assoc, 1979, P 175 Linford E, Glover FA, Bishop C, et al: The relationship between semen evaluation methods and fertility in the bull. J Reprod Fertil47:283-29, 1976 Lovelock JE: The hemolysis of human red blood cells by freezing and thawing. Biochem Biophys Acta 10:414, 1953 MacMillan KL, Watson JD: Fertility differences between groups of sires relative to stage of oestrus at the time of insemination. Anim Prod 21:243-249, 1975 Mazur P: Fundamental aspects of the freezing of cells with emphasis on mammalian ova and embryos. In Proc 9th Internat Cong Anim Reprod Artif Insem, vol 1, Madrid, 1980, p 99 Meryman HT: Mechanics of freezing in living cells and tissues. Science 124:515, 1956 Oltenacu EAB, Foote RH: Monitoring fertility of AI programs: can nonreturn rate do the job? Proc 6th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1976, p 61 Pace MM, Sullivan JJ: A biological comparison of the .5 mL ampule and .5 mL French straw systems for packaging bovine spermatozoa. Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, pp 3-9 Pellissier CL: Herd breeding problems and their consequences. J Dairy Sci 55:385391, 1972 Pellissier CL: Dairy cattle breeding problems and their consequences. Theriogenology 6:575-583, 1976 Peters JL, Senger PL: Radiographic method for evaluation of bovine artificial inseminating technique. J Dairy Sci 66:1760-1764, 1983 Pickett BW: Field techniques and semen handling. In Proc 1st Tech Conf Artif Insem Bovine Reprod, 1966, p 64 Pickett BW: Factors affecting the utilization of frozen bovine semen for maximum reproductive efficiency. AI Digest 19:8, 1971 Pickett BW, Martig RC, Cowan WA: Preservation of bovine spermatozoa at -79 and -196°C. J Dairy Sci 44:2089, 1961 Rapatz G: What happens when semen is frozen. In Proc 1st Tech Conf Artif Insem Bovine Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1966, p 45 Robbins RK, Sullivan JJ, Pace MM, et al: Timing insemination of beef cattle. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia Mo, 1978, p 97 Saake RG: Concepts in semen packaging and use. In Proc 8th Conf Artif Insem Beef Cattle, 1974, p 11 Saake RG: Factors affecting spermatozoan viability from collection to use. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, p 39 Saake RG: Semen quality, importance of and influencing factors. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1984, p 30 Saake RG, Marshall CE, Vinson WE, et al: Semen quality and heterospermic
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insemination in cattle. In 9th Int Cong Anim Reprod Artif Insem, vol 5, Madrid, 1980, pp 75-78 Salisbury GW, Vandemark NL: As quality increases, fertility increases until optimum female population is reached. In Physiology of Reproduction and Artificial Insemination of Cattle. San Francisco, WH Freeman and Co, 1961 Schalns 0, Schallenberger E, Hoffmann B, et al: The oestrous cycle of the cow: Hormonal parameters and time relationships concerning oestrus, ovulation, and electrical resistance of the vaginal mucus. Acta EndocrinoI86:180-192, 1977 Seguin B: Technique factors influencing pregnancy rates. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeeders, Columbia, Mo, 1984, p 122-125 Senger PL, Hillers JK: On the farm management of semen tanks. In Proc 8th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1980, pp 25-29 Senger PL, Hillers JK, Mitchell JR, et al: Effects of serum treated semen, bulls and herdsmen inseminators on conception to first service in large commercial dairy herds. J Dairy Sci 67:686-692, 1984 Senger PL, Peters JL, O'Connor ML: Radiographic evaluation of insemination technique. A training and research tool. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1984, p 114-121 Spalding RW, Everett RW, Foote RH: Fertility in New York artifically inseminated Holstein herds in dairy herd improvement. J Dairy Sci 58:718-723, 1975 Trimberger GW: Breeding efficiency in dairy cattle from artificial insemination at various intervals before and after ovulation. Neb Ag Exp Stat Res Bull 153, 1948 Van Duijn C Jr: Relationships between characteristics of spermatozoa and fertility. Ann BioI Anim Biochem Biophys 5:419-443, 1965 Zemjanis R, Fahning ML, Schultz RH: Anestrus, the practitioner's dilemma. Vet Scope 14:15, 1969
Address reprint requests to Albert D. Barth, DVM, M Vet Sci Department of Herd Medicine and Theriogenology Western College of Veterinary Medicine University of Saskatchewan Saskatoon, Saskatchewan S7N OWO Canada
0749-0720/93 $0.00
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FACTORS AFFECTING FERTILITY WITH ARTIFICIAL INSEMINATION Albert D. Barth, DVM, M Vet Sci
The reproductive performance of individual beef or dairy herds is extremely important and plays a major role in the financial success of livestock enterprises. Economically, the most important reproductive goal of beef and dairy herds is that each cow must calve on a yearly basis. Most beef cows are managed on a seasonal basis and must calve every 12 months in a defined calving season. Failing to do so leads to a nonpregnant status or an expected late calving date and such cows must be culled to maintain herd reproductive efficiency. Most commercial beef cattle and a large proportion of purebred beef cattle are bred naturally, and the use of highly fertile bulls is of utmost importance. A significant number of beef cattle, particularly in purebred herds, however, are bred by artificial insemination (AI). Cows often are given one chance to become pregnant via AI and then are exposed to "clean up" bulls for the remainder of the breeding season. Success with AI in such herds is highly dependent on accurate heat detection, use of highquality semen, and proper insemination technique. In North America, most dairy cows are not managed to freshen on a seasonal basis, but to maintain a high level of productivity, dairy cows also must calve yearly. Most dairy herds would find a goal of a 12-month calving interval difficult to achieve, however, and the economics of such a goal may be questioned as well. Some high producing dairy cows may be most productive with a somewhat longer calving interval; a 12.5- to 13-month herd calving interval may be a more realistic goal. Unlike beef cattle, most dairy cows are bred by AI. Low From the Department of Herd Medicine and Theriogenology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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breeding efficiency is one of the most serious economic problems confronting dairy management. 26, 27 When conception is delayed and the calving interval is lengthened, milk revenue usually is reduced because cows spend a greater portion of their lactation at low production levels. Dairy and beef cows that fail to conceive or that conceive too late to justify their retention in the herd must be sold for slaughter, often at a much lower value than their purchase price or rearing cost. The costs of AI increase in proportion to the additional breedings required, and the cost of veterinary services and medication in repeat breeding cows also may be increased. Additional losses due to delayed genetic progress can be substantial, although impossible to calculate. Breeding problems tend to discourage the use of bulls with a high predicted difference that demand a premium price for semen, particularly for repeat services. Cows with low productivity must be culled to maintain economic efficiency, but this culling potential is greatly reduced in herds with breeding problems because of the number of cows that must be sold due to breeding failure and because fewer replacements are born. In general, any herd consists of three kinds of cows, classified according to reproductive status-those that are pregnant, those that have calved within the past 45 days (the period for uterine involution and resumption of estrous cyclicity), and those that must be rebred for the next gestation. Every cow in the herd falls into one of these three categories. Cows in the first two categories cannot contribute to the herd's current reproductive problems. If a yearly calving interval is desired, then cows with a 285-day gestation period and a 45-day postpartum period have 35 to 50 days in which to become pregnant. It is obvious that an excessive number of days in the breeding period is incompatible with a high reproductive rate. The average number of days open for all cows in the herd therefore is a good measure of the current reproductive status of the herd. There are many reasons for failure of cows to become pregnant and an excessive accumulation of days open." The two most important are cows that are not bred and cows that must be rebred. Organic conditions such as uterine infections, cystic ovaries, and abortions generally make only minor contributions to days open. More commonly, reproductive problems in a herd are attributable to management problems. Lack of ovarian cyclicity or low conception rate in cycling dairy cows because of high milk production and a negative energy balance are important herd management problems, but as many as 90% of anestrus cases are due to failure in heat detection. 47 With good management, AI can be highly successful if semen is placed into the cow's uterus near the time of ovulation. In this regard, efficient, accurate heat detection is of paramount importance. In addition, because conception rates are low when inseminations are done improperly, proper handling of semen and proper insemination technique are equally important. JI
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THE STATUS OF ARTIFICIAL INSEMINATION IN NORTH AMERICA
Artificial insemination offers several distinct advantages over use of natural mating, and this has resulted in the development of a highly successful AI industry. The use of AI on a commercial basis developed quickly after World War II. Most semen initially was chilled liquid semen with a short life span. One of the major advantages of AI is to extend the use of superior sires over a higher number of females than would be possible with natural service. With the advent of storage of frozen semen in the 1950s, sire testing programs became more feasible, allowing for more intensive genetic selection and rapid genetic improvement. Other potential advantages of AI include the elimination of bulls with fertility problems and bulls with unexpected variations in fertility. Strictly controlled health regulations of AI bulls have prevented disease transmission between females, which occurs more readily when natural service is used. Artificial insemination also has the advantage of improved safety on farms through the elimination of bulls, particularly dairy bulls, which frequently caused injury and death. The percentage of cattle bred by AI in North America varies among regions but includes approximately 75% of dairy cows and 5% of beef cows on a national basis for both the United States and Canada (Canadian Association of Animal Breeders, and National Association of Animal Breeders, personal communication, 1992). The use of AI in the beef industry has developed slowly and is still limited in commercial herds, but it now is used fairly extensively in purebred herds. In the past 10 to 15 years, there has been a significant increase in the number of breeders storing frozen semen on the farm and breeding their own cows. A very substantial portion of the cows are still inseminated by professionals employed by AI organizations, however. These organizations also provide training courses in insemination and provide semen sales and delivery to the farm. The AI industry has worked diligently to improve cryopreservation technology and AI centers strive to provide a high quality product that will perform well if used correctly. When healthy fertile cows are inseminated with frozen semen from highly fertile bulls at the correct time in their estrous cycle and the semen is properly placed into the uterine body, pregnancy rates similar to those seen with natural service can be expected. In three different studies2, 8, 14 in which heifers were slaughtered 3 to 5 days after AI, the fertilization rate was 90% to 100%. However, AI is a technical procedure under human influence and many factors may adversely affect pregnancy rates obtained by AI. Calving rates are substantially lower than fertilization rates because cows are not always in excellent health or mated at the proper time and not all conceptuses become established in the uterus or grow normally to parturition. In general, AI centers today average 60- to 90-day nonreturn rates of about 70%. The nonreturn rates, when calculated for large numbers of inseminations, are reliable measures for comparison of relative fertility among individual bulls or inseminators, but they
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substantially overestimate the true calving rates. In a Minnesota study, conception rates for 2270 services in 17 dairy herds during 1980 and 2217 services in 18 dairy herds in 1982 and 1983 were 41% and 39% respectively.40 Studies in New York and California have both shown that the true calving rates to first services were around 50%, even though the AI centers routinely reported 60- to 90-day nonreturn rates of 65% to 75%. Pellissier27 reported that the conception rate from 12,964 first services in California dairy herds was 44.2%. In the same study, only 57.7% of the cows conceived in the usually accepted optimum 61to 120-day postpartum period. In a New York survey, data from 9750 cows in 125 dairy herds on Dairy Herd Improvement records showed a 50% pregnancy rate after one service compared with a 58% nonreturn rate. 44 The yearly average 60- to 90-day nonreturn rate in herds using the same AI service was 69%. The two principal factors identified in this study accounting for the difference between actual conception and 60- to 90-day nonreturn rate were cows that died or were disposed after a first service and never received a second service and cows that were presented for a second service beyond the 60- to 90-day postbreeding interval.
EFFECT OF TIME OF INSEMINATION ON PREGNANCY RATE ON FERTILITY
The general recommendation has been to breed cows in the middle to the end of standing .heat for optimum fertility. Because the period of estrus may vary from 6 to 24 hours, however, it is difficult to determine when the midpoint is reached. The general guideline for determining insemination time originated in a 1948 study by Trimberger,45 in the form of the AM-PM rule-i.e., if cows are first observed in heat in the morning (AM) they should be bred that afternoon (PM); if they are first seen in heat in the late afternoon (PM), they should be bred the next morning (AM). In the Trimberger study, the time of breeding was divided into nine different time periods with 16 cows per breeding period. Five time groups were used to separate the 24 hours prior to ovulation. The overall conception rate was 69.4% (50172) and the best time for breeding was 13 to 18 hours prior to ovulation (12/14 for an 85.7% pregnancy rate). The time relationships concerning estrus, luteinizing hormone (LH) release, and ovulation in 61 heifers and cows were reported by Schams et al in 1977. 39 In their study, the surge of LH peaked at 6.4 ± 3 hours after the onset of estrus and ovulation occurred at 25.7 ± 6.9 hours after the LH peak. Their data also indicated that, although the preovulatory LH peak occurred at any time during the day, it peaked predominantly at around 8 AM or 8 PM. As a result, there were two main periods for ovulation. According to these data, insemination at 12 hours after the onset of estrus would result in insemination at about 20 hours prior to ovulation. This agrees fairly well with the AM-PM rule, which would result in insemination at the
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optimum time suggested by the Trimberger study of approximately 13 to 18 hours prior to ovulation. Although one study 17 found that conception rates in 367 heifers were higher when breeding occurred in 2 to 6 hours after onset of estrus than when inseminations took place 10 or more hours after the onset of estrus, other recent studies have supported the AM-PM rule. MacMillan and Watson,21 in New Zealand, examined over 6000 inseminations to determine the effects of stage of estrus at the time of breeding on conception rates. Inseminations were done in the morning with unfrozen extended semen collected the previous day using 2.5 million total cells per dose. The mean pregnancy rates for all sires, as determined by 18- to 49-day nonreturn rates, were 65.7%, 69.3%, 75.1%, and 72.8%, respectively for breedings made in early, mid, and late estrus or postestrus. For sires of above average fertility, nonreturn rates for the different insemination times tended to be higher for late estrus inseminations but were not statistically different. For bulls of average or low fertility, nonreturn rates for the four insemination times differed significantly. In another study, 12 in which dairy cows were inseminated either immediately after observed estrus (358 inseminations) or 12 hours after observed estrus (360 inseminations), the pregnancy rates were 51.1% and 55% for O-hour and 12hour breedings, respectively, but the difference in pregnancy rate was not statistically significant (P> .05). In a study involving 2091 first inseminations in beef cows on range, pregnancy rates were compared for successive 2-hour periods from estrus detection to insemination. The overall first insemination calving rate was 63% for cows inseminated between 12 and 29 hours after estrus detection. The highest pregnancy rates occurred for inseminations done 16 to 25 hours after estrus detection. 33 From these studies it would appear that the best time for insemination in the cow is 12 to 20 hours after the first observation (onset) of estrus. When heat detection is done in the morning and evening, therefore, the AM-PM rule established by Trimberger would be appropriate.
THE EFFECT OF VARIATION IN SEMEN QUALITY ON FERTILITY
Considerable research information has been generated over the past 40 years on all aspects of cryopreservation technology and semen quality requirements for successful AI in cattle. Although industry standards for semen quality are not uniform and are not regulated, the commercial viability of AI centers depends on the production of a highquality product that performs well in the hands of the consumer. Artificial Insemination centers have been leaders in research and development and have built a highly reputable industry. Nevertheless, AI centers, particularly those that derive a great deal of their revenues from custom collection of beef bulls, often are under a great deal of pressure to preserve semen of marginal quality. In many cases, cattle
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breeders have invested a great deal of money in a bull and must sell semen as quickly as possible to recover their investment. Many young beef bulls are still undergoing puberty and are still producing lowquality semen when cryopreservation is begun. These young beef bulls, as well as older beef bulls entering AI centers, often have been overfed and have been under considerable stress associated with the show circuit. They often require several months to adjust to the new environment of an AI center and need to lose body weight and fat from the scrotum before they are capable of producing good-quality semen. However, cattle breeders often are adamant that their bulls must maintain or gain weight during semen collection because high body weight generally is seen as a reflection of the growthiness of a bull-a desirable trait that it is hoped he would pass on to his offspring. Beef bulls frequently are selected for growth rate, height, coat color patterns, and other physical characteristics not related to fertility. Although these bulls may be show winners and bring the highest prices at prestigious sales, they may have abnormalities such as testicular hypoplasia, epididymal anomalies, and hereditary sperm defects that preclude them from ever being able to produce good semen. Furthermore, many beef cattle breeders are willing to accept a reduced level of semen performance in order to obtain the desired genetics from a popular bull. Thus, there is a ready market for a certain amount of semen that is of marginal quality. Most dairy bulls are owned by AI centers and not subject to many of the reproductively detrimental influences experienced by beef bulls destined for AI. In general, semen from dairy bulls is less likely to be of marginal quality than that of beef bulls. Those involved in the embryo transfer industry must be aware that semen that results in lower but perhaps acceptable fertility in single ovulating females usually performs more poorly when used in superovulated donor cows. Because of the high costs involved in embryo collection and transfer, only semen of high fertility should be used, and it would seem prudent to evaluate each freeze batch of semen prior to superovulation of the donor cow. The validity of laboratory assays for prediction of fertility of frozen semen often is questioned, and correlations of specific semen traits with fertility have ranged from very low to very high. 5- 7, 9, 19, 46 One therefore might conclude that little is known about semen quality as it relates to fertility. However, the inability to obtain high correlations between semen quality traits and fertility does not necessarily mean that semen evaluations lack validity. Indeed, many experiments that were unable to demonstrate a correlation between semen quality and fertility lacked validity themselves. In general, the reasons for low correlations between semen quality traits and fertility rest largely in the inability of experiments to account for experimental error. Some examples include: 1. The assessment of the effect of single traits of semen on fertility does not take into account variability caused by other traits. The effects of differences in percentage of progressively motile spermatozoa on fertility, for example, may be masked by unaccounted for variation in
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sperm morphology or sperm dose. When these sources of error are not accounted for, differences in fertility due to a trait such as sperm motility are minimized and resulting correlations are low. 2. There may be insufficient difference between the quality traits under assessment to result in a difference in fertility. As the quality of semen increases, the resultant fertility increases until the highest fertility potential of the female population is reached. Further increases in semen quality would not result in any further increase in fertility. 38 When all differing levels of semen quality traits being assessed are adequate for optimum fertility in the female population, therefore, a low correlation for the trait measured results. Indeed, in many of the reported fertility trials, the semen that was compared with control semen had not been rejected because it had met the minimum quality requirements of the AI center. Linford et aP9 demonstrated the importance of using semen with large differences in quality for demonstrating correlations between laboratory tests and fertility. When bulls with relatively low variation in fertility were used, correlations between semen quality tests and fertility were low. When a second group of bulls, in which there was a larger variance in semen quality, was used, correlations between semen quality tests and fertility were greatly improved. 3. The most commonly used measure of fertility in semen quality research has been the nonreturn rate (NRR) of cows to estrus after insemination. Before the days of direct semen sales and owner-producer inseminators, each AI center had a quick and inexpensive way of retrieving information on semen performance in the field. The NRR, however, has some inherent sources of error that are difficult to control. Recordkeeping and reporting often are not strong points in the management of herds. Many producers use a herd bull for some of the breeding and bulls have always had the notoriety of unexpectedly producing calves from cows thought to be pregnant via AI. Cows that do not conceive after one or more inseminations may be culled but would appear in the NRR data not to have returned to heat. Similarly, cows that return to heat a second or third time may be rebred with semen from different AI centers. Each center would receive receipts for only one breeding and assume a first-service conception. Heat detection efficiency often does not exceed 50%. Hoffmann et al13 reported that cows that show no behavioral estrus after insemination although they are not pregnant, and those inseminated during the luteal phase (and thus "not returning to estrus") comprise between 20% and 27% of cows inseminated. Most NRR data are obtained for the 60- to 90-day period after insemination(s). The difference between conception rate and NRR increases as the number of inseminations per cow increases. This difference was reported to be 15.9% for cows receiving a third service. 24 Therefore, although the NRR system of estimating fertility allows ranking of the relative fertility of bulls, it is inherently inaccurate as a measure of conception rate. Furthermore, it has a tendency to enhance the apparent fertility of low fertility semen, thereby reducing the apparent variance between good and poor semen. As a result, the
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correlation between semen quality traits and fertility measured by the NRR appears lower than it actually may be. Saake36 used heterospermic insemination (competitive fertilization) to investigate the validity of laboratory tests of semen quality to predict the fertility of frozen semen. Heterospermic insemination refers to the use of sperm from more than one male in an insemination dose. Semen usually is mixed such that sperm numbers from each competing male are equal and it is presumed that paternity of offspring should also be equal unless there is a difference in semen quality among competing males. The ratio of offspring based on paternity is converted to a competitive index. It has been shown that males that are highly fertile homospermically (high fertility when used under normal circumstances) also are superior heterospermically. 3 Large differences among males detected heterospermically, however, frequently are very small or not apparent when these bulls are tested homospermically. In addition to the improved variance in fertilizing potential offered by heterospermic insemination, this technique also minimizes or eliminates the masking influences of variation in female fertility and other biologic or environmental factors that may prevail at the time of insemination. Saake et al,36 using frozen bovine semen, obtained relatively high correlations between eight different semen quality measurements and competitive indexes from heterospermic inseminations. It has long been recognized that the repeatability of semen quality tests is not high among laboratories and even within laboratories. 11 This is particularly true for subjective assays. The use of computer assisted semen analysis has allowed objective assessment of semen quality traits and should improve repeatability, but the cost of the equipment may be out of reach or unjustified economically for many enterprises. At the present, therefore, we must continue to rely on subjective assessment of semen quality.
THE EFFECT OF SEMEN HANDLING ON FERTILITY
One of the major causes of low fertility associated with AI is mishandling of semen. This fact has been confirmed by studies involving professional inseminators closely supervised by AI organizations. 29-31 It seems likely that this problem would be accentuated on the farm and ranch by herd-owner inseminators who often have minimal training and no supervision. Over time, even professional inseminators may develop bad habits and a relaxed attitude toward semen handling and insemination technique, resulting in declining fertility. Several studies indicate the detrimental effects of exposure of frozen semen to ambient temperatures. The key factor to long-term cryopreservation is the very low (-196°C) temperature of the liquid nitrogen (LN) tank. Many do not realize the extreme importance of keeping exposures of semen to ambient conditions to an absolute minimum. From available evidence,
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it appears that temperatures should be maintained at -130°C or lower at all times. 16, 23, 32 Above this critical temperature, a phenomenon called recrystallization may occur, resulting in damage to cellular structures. During the freezing process, pure water is removed from solution in the formation of ice crystals and solutions increase in concentration. The resulting changes in osmotic pressures remove most of the intracellular water from spermatozoa or embryos. This dehydration occurs early during the freezing process and is essential to survival of cells during cryopreservation. Because of the rapid rate of freezing of spermatozoa, the remaining intracellular water forms very small ice crystals that appear to be relatively harmless. At temperatures above - 130°C, however, molecules of WOlter may leave ice crystals and reattach to other crystals, resulting in an increase in the size of some crystals at the expense of others (recrystallization). The larger crystals are potentially damaging to cellular structures. 32 Another source of damage, which occurs at intermediate temperatures above - BO°C, is the highly concentrated solution (solution effect) that results during the freezing and thawing process. 20, 22, 23 Injury is believed to occur by salt solution denaturation of specific enzymes and cell membranes, or loss of structural integrity when the cell shrinks below a minimum volume. 1 The relatively large surface area and small volume of straws used in cryopreservation are conducive to rapid heat exchange. Spermatozoa in 0.2S-mL straws are particularly vulnerable to rapid rises in temperatures upon exposure to ambient conditions. In this regard, semen that formerly was predominantly packaged in I-mL glass ampules had a much larger margin of safely than straws during handling in field situations. This was illustrated in the work of Berndtson et al, 4 which showed that 0.2S-mL and O.S-mL straws held by forceps and exposed to ambient air temperatures of 20 ± 0.6°C reached the temperature of recrystallization within 10 to 15 seconds. Under similar conditions, 1mL ampules clipped to metal canes required approximately 45 seconds to reach the temperature of recrystallization. Under field conditions, rates of warming upon exposure to ambient conditions may be much higher because of the effects of wind or sunshine. It is important to recognize that damage due to recrystallization is cumulative. Damage due to initial exposure may not be overt, but after repeated exposures, a reduction in post-thaw viability may become evident. Incubation of semen for 2 to 4 hours post-thaw may reveal such damage more clearly than immediate post-thaw examinations. Clearly, individual straws should never be exposed to ambient temperatures. Straws within goblets, particularly when the level of LN in the tank is high enough to fill the goblets, are much less susceptible to rapid temperature changes during removal of straws for AI or when transfers are made to other tanks. This was illustrated in the work of Berndtson et al, 4 which showed that when 10 0.2S-mL or 5 O.S-mL French straws per goblet were exposed to 20 ± 0.6°C ambient conditions, the temperature of recrystallization was not reached until 45 to 60 seconds of exposure.
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The danger of warming of semen occurs not only with exposure of straws to ambient conditions external to the LN tank. Exposure to potentially damaging temperatures occurs each time straws are raised to the neck of a tank to permit removal of an individual straw for thawing. In a report by Berndtson et al,4 the temperature within the neck of a LN semen storage tank was ranged from approximately - 180°C to + 2°C. Saake37 showed that the rate of warming to - 80°C was virtually identical for O.S-mL straws exposed to either - 22°C or + SoC temperatures. Therefore, canisters should be raised no higher within the neck of the LN tank than absolutely necessary and the individual straw should be removed as quickly as possible. In some instances, when large numbers of inseminations or embryo transfers are done sequentially, straws are repeatedly exposed to ambient temperatures. Unless sufficient time is allowed between exposures to allow cooling to - 196°C, higher temperatures will be reached during each exposure. When semen in straws placed in goblets on metal canes was raised to the top of the canister neck for five consecutive I-minute exposures, allowing I-minute returns to LN vapor between exposures, more than 10 minutes were required for straw temperatures to return to - 196°C after the fifth exposure. Ideally, the level of LN in the storage tank should be high enough so that the semen would be plunged back into the LN upon each return to the tank. 37 The foregoing studies suggest that semen stored and used in the field may be subject to gradual deterioration. In one study,25 test semen was placed in 39 LN field tanks and, over a period of 6 months, the semen was given at least 480 exposures to ambient conditions. When compared with those stored at the AI center, I-mL glass ampules showed no difference in viability of spermatozoa stored under field conditions. Significant deterioration in semen viability was found in semen packaged in O.S-mL straws, however. This difference was seen more readily after semen was incubated at 37°C for 2 to 4 hours. In addition, spermatozoa located in the top goblet had a significantly lower percent intact acrosomes than those in the bottom goblet. This study demonstrated that deterioration of semen in field units is quite possible. It is unlikely, however, that semen in field use would be exposed to the neck of the tank as frequently as in the foregoing study. In two subsequent studies,18, 41 test semen was placed in 60 semen tanks belonging to cooperating dairy producers. Semen was removed at 6-, 12-, 18-, and 24-month intervals and compared to semen of similar freeze batches that were stored at the AI center. In these studies, no difference was found in semen viability between on-farm and centrally stored semen. Furthermore, no difference in viability was seen between semen from top or bottom locations on the canes. This indicates that semen handled carefully in a routine farm situation should suffer no adverse effects. American Breeders Service in Colorado has developed a system to indicate whether temperatures within a tank have been raised to dangerous levels. Two ampules, each containing either blue or red liquid, frozen into the apex of the ampule are placed on a cane that is
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then placed with frozen semen in the LN tank. If temperatures rise sufficiently to thaw the colored fluid, it sinks to the bottom of the ampule. If only the blue ampule thaws, damage to the semen may not be very severe, but if both ampules thaw (blue and red fluid sinks to the bottom of the ampules), semen must be tested before use. The materials for this method of monitoring LN tank temperature are commercially available from Minitube of America Inc., Madison, Wisconsin. Sudden unexpected loss of LN from a tank containing embryos or semen may result in an economically disastrous loss. One report indicated that the failure rate of LN tanks in 57 dairy herds was approximately 5% over a 2-year period. 41 Liquid nitrogen tanks are expected to maintain a vacuum for at least 15 to 20 years if they are not abused, but it is not uncommon for new tanks to lose their vacuum even without any evidence of damage to the tank. There may be an indication that a tank is losing its vacuum when it seems to lose nitrogen at a faster rate than indicated by its specifications or when it needs to be replenished more frequently than usual; most tank failure is rather sudden, however. When vacuum is lost, all of the nitrogen may be lost from the tank within 24 hours. Rapid evaporation of nitrogen results in pronounced cooling of the top portion of the neck and this results in condensation of moisture from the atmosphere at the top of the tank. Depending on the rate of loss and whether the loss is detected while there is still LN in the tank, there will be a small build-up of frost or moisture at the top of the neck. Once all the nitrogen has evaporated, the neck warms up, the frost and moisture evaporate, and the tank looks like a normal tank. Usually, only the lid attached to the styrofoam neck piece becomes frosty or wet. The amount of frost or moisture may be very small, showing only light frosting on the parts of the lid touching the handles of the canisters below. New tanks and tanks that have been moved or shipped are more likely to suddenly lose vacuum than static holding tanks, but even static tanks have been known to lose vacuum suddenly. All tanks therefore must be monitored closely. Perhaps the only practical means of preventing loss of semen is to attach LN level sensors with warning indicators to every tank. Another means to ensure the preservation of at least half the semen is to divide it among two or more tanks, so if one tank fails, not everything is lost.
THE EFFECT OF INSEMINATOR EXPERTISE ON FERTILITY
In order to achieve optimum fertility with AI, inseminators must be able to place the semen into the uterus of the cow quickly and with a minimum of trauma to the cervix and endometrium. The junction of the cervix and the uterine body usually is recommended as the target for semen deposition in AI. This allows the semen to bypass the cervical
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barrier and should allow an equal number of sperm to enter each uterine horn. Thus, equal pregnancy rates are anticipated between the sides of the tract regardless which ovary contains the ovulatory follicle. Experiments have been done to determine whether better conception rates could be achieved by placing half the inseminate into each uterine horn (horn breeding) or by placing the inseminate on the side of ovulation rather than within the uterine body. In one study,40 the conception rate for 114 horn-bred cows was 67% versus 64% in 110 cows inseminated into the uterine body. In the same study, the conception rate in 44 cows and heifers inseminated on the side of ovulation was 70% compared with 75% for cows and heifers inseminated into the uterine body. The differences in fertility related to location of semen deposition were not significant, so, given that it is easier to deposit semen into the uterine body than up the uterine horn, the uterine body remains the best target site for semen deposition. Even so, previous work with dye testing showed that the AI target frequently is missed. 10, 15 The expertise of inseminators may vary according to the amount of training and practice as well as personal qualities that may influence the quality of work performed. In a study of 2820 first services, marked differences were reported in the performance of herdsmen inseminators.42 In this study, 10 proven AI bulls were used in four large commercial dairy herds. Other variables that might affect conception rates were controlled experimentally or accounted for statistically. Conception rate was determined by rectal palpation 35 to 45 days after insemination. The inseminator with the highest rate achieved a first-service conception rate of 63%, whereas the inseminator with the lowest rate achieved only 39%. Radiography has been used to visualize the interior of excised bovine reproductive tracts from packing plants to assess accuracy of semen placement. With this technique, reproductive tracts were placed in a device that simulated the anatomic position of the reproductive organs in vivo. Air was introduced to provide contrast between the uterine lumen and tissue and semen extender was treated with lead acetate to provide radiographic opacity of the inseminate. 28 This technique was used to evaluate and compare the insemination techniques of 20 professional and 20 herdsmen inseminators.43 Each inseminated 20 tracts. There was no difference in the ability of professionals and herdsmen inseminators to position the syringe tip in the uterine body. There was significant variation in correct syringe tip placement between individuals, however. Correct placement was achieved in 6% to 85% of cases by professionals and in 0 to 82% of cases by herdsmen inseminators. Fewer than half (39%) of placement attempts by all inseminators were in the uterine body. In 36% of placement attempts, the syringe tip was positioned in either the left or right uterine horn. The mean percentage of semen deposits within the cervix was 13% by professionals and 21 % by herdsmen inseminators. It would appear that marked improvements to AI fertility could be achieved through improved insemination technique. In recent times, with the use of minimal numbers of spermatozoa per insemination
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dose and smaller insemination volumes due to the introduction of 0.25mL straws, it is of utmost importance that semen be placed properly within the uterus. There are no routine testing or retraining programs for inseminators in the United States or Canada. In light of the results of studies such as the ones just mentioned,43 it appears that a great many herdsmen and professional technicians would benefit from routine retraining programs.
SUMMARY
Man's intervention in the natural processes of reproduction with the use of AI has allowed rapid genetic improvement in beef and dairy herds and has resulted in a marked increase in livestock productivity. In today's tough economic climate, high reproductive efficiency is of utmost importance for livestock enterprises to remain viable. Many factors affect the success of AI programs; of particular importance are the health and nutritional management of the herd and accurate, efficient heat detection. Good management combined with knowledge, technical expertise and careful attention to detail in the timing of insemination in relation to the period of estrus, semen handling, and correct semen placement in the uterus ensure the successful use of AI.
References 1. Arakawa T, Carpenter JF, Kita AK, et al: The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27:401-415, 1990 2. Bearden HJ, Hansel WM, Bratton RW: Fertilization and embryonic mortality rates of bulls with histories of either low or high fertility in artificial breeding. J Dairy Sci 39:312-318, 1956 3. Beatty RA, Bennett GH, Hall JG, et al: An experiment with heterospermic insemination in cattle. J Reprod Fertil 19:491, 1969 4. Bemdtson WE, Pickett BW, Rugg CD: Procedures for field handling of bovine semen in plastic straws. In Proc 6th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1976, pp 51-60 5. Bishop WH, Campbell RC, Hancock JL, et al: Semen characteristics and fertility in the bull. J Agric Sci 44:227-248, 1954 6. Bratton RW, Foote RH, Henderson CR, et al: The relative usefulness of combinations of laboratory tests for predicting the fertility of bovine semen. J Dairy Sci 34:1542, 1956 7. Buchner PJ, Willet EL, Bayley N: Laboratory tests singly and in combination for evaluating fertility of semen and of bulls. J Dairy Sci 37:1050, 1954 8. Diskin MG, Sreenen, JM: Fertilization and embryonic mortality rates in beef heifers after artificial insemination. J Reprod Fert 59:463-468, 1980 9. Elliott PI: Significance of semen quality. In Salisbury W, VanDemark NL, Lodge JR (eds): Physiology of Reproduction and Artificial Insemination of Cattle, ed 2. San Francisco, WH Freeman and Co, 1979, pp 428-441 10. Graham EF: The use of a dye indicator for testing, training and evaluating technicians in artificial insemination. In Proc 1st Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1966, pp 57-60 11. Graham EF, Schmehl MKL, Nelson DS: Problems with laboratory assays. In Proc 8th
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Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1980, pp 59-66 Gwasdauskass FC: The effect of time of insemination on conception rates in dairy herds. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, pp 92-96 Hoffmann B, Gunzler 0, Hamburger R, et al: Milk progesterone as a barometer for fertility control in cattle. Methodological approaches and present status of application in Germany. Br Vet J 132:469, 1976 Kidder HE, Black WG, Wiltbank IN, et al: Fertilization rates and embryonic death rates in cows bred to bulls of different levels of fertility. J Dairy Sci 37:691-697, 1954 King GJ, MacPherson JW: Observations on retraining artificial insemination technicians and its importance in maintaining efficiency. Can Vet J 6:83-85, 1965 Larson GL, Graham EF: Effects of low temperatures in storage of bovine semen. AI Digest 6:6, 1958 Laster DB, Glimp HA, Gregory KE: Age and weight at puberty and conception in different breeds and breed crosses of beef heifers. J Anim Sci 34:1031-1036, 1971 Lineweaver JA, Saake RG, Gwasdauskas FC: Effect of double decking and storage time on semen stored in farm storage tanks. In Proc Am Dairy Sci Assoc, 1979, P 175 Linford E, Glover FA, Bishop C, et al: The relationship between semen evaluation methods and fertility in the bull. J Reprod Fertil47:283-29, 1976 Lovelock JE: The hemolysis of human red blood cells by freezing and thawing. Biochem Biophys Acta 10:414, 1953 MacMillan KL, Watson JD: Fertility differences between groups of sires relative to stage of oestrus at the time of insemination. Anim Prod 21:243-249, 1975 Mazur P: Fundamental aspects of the freezing of cells with emphasis on mammalian ova and embryos. In Proc 9th Internat Cong Anim Reprod Artif Insem, vol 1, Madrid, 1980, p 99 Meryman HT: Mechanics of freezing in living cells and tissues. Science 124:515, 1956 Oltenacu EAB, Foote RH: Monitoring fertility of AI programs: can nonreturn rate do the job? Proc 6th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1976, p 61 Pace MM, Sullivan JJ: A biological comparison of the .5 mL ampule and .5 mL French straw systems for packaging bovine spermatozoa. Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, pp 3-9 Pellissier CL: Herd breeding problems and their consequences. J Dairy Sci 55:385391, 1972 Pellissier CL: Dairy cattle breeding problems and their consequences. Theriogenology 6:575-583, 1976 Peters JL, Senger PL: Radiographic method for evaluation of bovine artificial inseminating technique. J Dairy Sci 66:1760-1764, 1983 Pickett BW: Field techniques and semen handling. In Proc 1st Tech Conf Artif Insem Bovine Reprod, 1966, p 64 Pickett BW: Factors affecting the utilization of frozen bovine semen for maximum reproductive efficiency. AI Digest 19:8, 1971 Pickett BW, Martig RC, Cowan WA: Preservation of bovine spermatozoa at -79 and -196°C. J Dairy Sci 44:2089, 1961 Rapatz G: What happens when semen is frozen. In Proc 1st Tech Conf Artif Insem Bovine Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1966, p 45 Robbins RK, Sullivan JJ, Pace MM, et al: Timing insemination of beef cattle. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia Mo, 1978, p 97 Saake RG: Concepts in semen packaging and use. In Proc 8th Conf Artif Insem Beef Cattle, 1974, p 11 Saake RG: Factors affecting spermatozoan viability from collection to use. In Proc 7th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1978, p 39 Saake RG: Semen quality, importance of and influencing factors. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1984, p 30 Saake RG, Marshall CE, Vinson WE, et al: Semen quality and heterospermic
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insemination in cattle. In 9th Int Cong Anim Reprod Artif Insem, vol 5, Madrid, 1980, pp 75-78 Salisbury GW, Vandemark NL: As quality increases, fertility increases until optimum female population is reached. In Physiology of Reproduction and Artificial Insemination of Cattle. San Francisco, WH Freeman and Co, 1961 Schalns 0, Schallenberger E, Hoffmann B, et al: The oestrous cycle of the cow: Hormonal parameters and time relationships concerning oestrus, ovulation, and electrical resistance of the vaginal mucus. Acta EndocrinoI86:180-192, 1977 Seguin B: Technique factors influencing pregnancy rates. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeeders, Columbia, Mo, 1984, p 122-125 Senger PL, Hillers JK: On the farm management of semen tanks. In Proc 8th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1980, pp 25-29 Senger PL, Hillers JK, Mitchell JR, et al: Effects of serum treated semen, bulls and herdsmen inseminators on conception to first service in large commercial dairy herds. J Dairy Sci 67:686-692, 1984 Senger PL, Peters JL, O'Connor ML: Radiographic evaluation of insemination technique. A training and research tool. In Proc 10th Tech Conf Artif Insem Reprod, Nat Assoc Anim Breeders, Columbia, Mo, 1984, p 114-121 Spalding RW, Everett RW, Foote RH: Fertility in New York artifically inseminated Holstein herds in dairy herd improvement. J Dairy Sci 58:718-723, 1975 Trimberger GW: Breeding efficiency in dairy cattle from artificial insemination at various intervals before and after ovulation. Neb Ag Exp Stat Res Bull 153, 1948 Van Duijn C Jr: Relationships between characteristics of spermatozoa and fertility. Ann BioI Anim Biochem Biophys 5:419-443, 1965 Zemjanis R, Fahning ML, Schultz RH: Anestrus, the practitioner's dilemma. Vet Scope 14:15, 1969
Address reprint requests to Albert D. Barth, DVM, M Vet Sci Department of Herd Medicine and Theriogenology Western College of Veterinary Medicine University of Saskatchewan Saskatoon, Saskatchewan S7N OWO Canada