- Email: [email protected]
Effect of prostaglandin F2α at insemination on sperm cell numbers and pregnancy rate in beef cattle
THERIOGENOLOGY
EFFECT OF PROSTAGLANDIN Flu AT INSEMINATION ON SPERM CELL NUMBERS AND PREGNANCY RATE IN BEEF CATTLE D. G. Morrison, 1,4 J. E2 Chandler,' L. S.3Chandler,* A. F. Radintz and M. B. Warner 1Rosepine Research Station and *Department of Dairy Science Louisiana State University Agricultural Center Baton Rouge, LA 70803 Received for publication: July 22, 1987 Accepted: March 22, 1988 ABSTRACT Fourteen cycling, nonlactating, multiparous beef cows were artificially inseminated (AI) 10 to 12 h after the onset of atural % estrus. One unit of frozen-thawed semen containing 100 x 10 total sperm cells was deposited into the body of the uterus. Immediately after AI, alternating cows were injected i.m. with either 25 mg (5 ml) of prostaglandin F20.(PGF)or 5 ml of 0.9% saline-benzyl alcohol control solution. Cows were slaughtered 16 + 1 h post AI, oviducts were retrieved, segmented into thirds (upper, middle and lower) and flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline. The number of sperm cells was counted using a phase contrast microscope. There were no right or left side effects (P=O.61) on the number of sperm cells recovered per oviduct within cow (389 vs 553; average SEM = 219). PGF had no effect (P=O.77) on the number of sperm cells recovered per oviduct (642 vs 300; average SEM = 231 for PGF and control females, respectively). More sperm cells were recovered from the lower third segment (P
prostaglandin, artificial insemination, sperm cells. pregnancy, beef cattle Acknowledgments 3 Former Associate, Idlewild Research Station, Clinton, LA 70722. 4 Reprint requests. Rosepine Research Station, P.O. Box 26, Rosepine, LA 70659. Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 87-92-1387.
JULY 1988 VOL. 30 NO. 1
109
THERIOGENOLOGY
INTRODUCTION Experimental treatments that improve sperm cell transport in laboratory animals may have relevance to problems of fertilization failure in larger animals. Fertilization failure in cattle is almost as.detrimental as embryonic mortality in reducing or delaying pregnancy (1,2). Prostaglandin F20(PGF) administration to rabbit does near the time of insemination has been reported to significantly increase the number of sperm cells in the oviducts 2.5 h after insemination (3). Higher ovum fertilization rates were also obtained when PGF was injected into does (4). When PGF was added to the inseminate or injected at the time of insemination of ewes, more sperm cells were recovered from the oviducts of treated than untreated ewes 16 or 24 h after insemination (5,6). PGF has been shown to not only increase contractility of the reproductive tract, thus increasing sperm cell transport, but also to reduce the normal loss of sperm cells from the reproductive tract, which is normally due to drainage or expulsion to the exterior. Thus more cells are available for transport to the upper tract (7). The effect of administration of PGF on sperm cell number and pregnancy rate has not beep assessed in cattle. The number of sperm cells in bovine oviducts has been shown to be highest between 8 and 16 h after insemination (8,9). The widespread use of AI in beef and dairy cattle makes the possibility of using an injectable compound to decrease services per pregnancy attractive. The objective of our investigation was to determine if PGF injection at the time of AI would increase the number of sperm cells .in the oviducts of cattle at about the time of ovulation and therefore increase the pregnancy rate.
MATERIALS AND METHODS Experiment 1 Fourteen Angus and Angus x Hereford, nonlactating, multiparous beef cows were used in this portion of the study. Ages ranged from 3 to 11 yr, and body condition was average to above average. All females had weaned at least one calf and were therefore considered to be fertile animals. Cows were observed for estrous activity during two consecutive estrous cycles to determine if cycles were typical in length (18 to 23 d) and if observable standing estrus was occurring. A surgically altered teaser bull wearing a chin-ball marking harness was kept with the cows as an aid in detecting estrus. Semen was collected fro% a mature'Jersey bull and packaged for freezing to contain 100 x 10 total sperm cells per 0.5 ml straw. Post thaw semen evaluation was performed as previously described (IO). Two units of semen were selected at random from each batch
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
for evaluation. Progressive motility, measured at 0 h post thaw and after 3 h of incubation at 37°C was 35 and 25X, respectively. Percentage of intact acrosomes and percentage of primary and secondary abnormalities were 65, 30 and 12%. respectively. Cows were artificially inseminated with a single unit of semen 10 to 12 h after being observed in estrus. Estrus observations were made at 0700 and 1800 h each day. Semen straws were thawed in a warm water bath (38'C) for 20 sec. Semen was deposited at the rim of the internal OS cervix and was therefore assumed to be in the body of the uterus. Immediately after AI (maximum of 1 min), alternate cows were injected i.m. with either 25 mg (5 ml) of PGF (Lutalyse, dinoprost tromethamine; The Upjohn Co., Kalamazoo, MI) or 5 ml of 0.9% saline-bensyl alcohol control solution. Females were transported approximately 12 km to a local slaughter facility and slaughtered 16 5 1 h after AI. As soon as possible (30 to 45 min), the reproductive tract was retrieved and the oviducts were ligated and excised. Examination of the ovaries revealed either a large (15 to 21 mm) follicle or an ovulation stigma for each cow. Oviducts were brought back to the laboratory where each oviduct (uterine tube) was sectioned into three equal The lower segment also segments (upper, middle and lower). included the utero-tubal junction and approximately 1 mm of the tip of the uterine horn. Each segment was flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline by the same technician (11). A minimum of two, lo-u1 smears of each flushing sample was counted. For each smear, the entire surface was scanned using a phase contrgst microscope (160X) to insure a count per 10 ul. Use of 100 x 10 sperm cells per insemination unit also helped to insure that sperm cells were available for6counting since this is five times the normal AI unit of 20 x 10 cells. Data were analyzed by a general linear models procedure (12). The model included the effects of treatment, side, ovulation, segment and all first order interactions. Treatment, side and segment were considered fixed effects, while ovulation was considered random. Cow within treatment by ovulation was used as the error term for testing treatment, ovulation and treatment by ovulation. Residual error was used for the other main effects and interactions. Experiment 2 One hundred and fourteen beef females (68 Angus x Hereford heifers and 46 Chianina crossbred postpartum suckled cows) were used to determine if PGF administered at AI would affect pregnancy rate. The two sets of females were maintained at different locations and inseminated by a single technician at each location. Surgically altered teaser bulls equipped with chin-ball marking devices were kept with the herd at all times as an aid in estrus detection. Cows were visually observed at least twice daily (0700 and 1800 h) for signs of estrus. Thgse observed in estrus were inseminated 10 to 12 h later with 20 x 10 motile sperm cells in 0.5~ml straws. Frozen
JULY 1988 VOL. 30 NO.’ 1
111
I’HERIOGENOLOGY
semen from a single Angus bull was used to inseminate the Angus x Hereford heifers. Semen from 16 Chianina sires was used at random to inseminate the Chianina cows. Semen was thawed as described in Experiment 1. Immediately after AI (maximum of 1 min), alternating females were administered PGF or vehicle i.m. as described above. Pregnancy'rates were determined by palpation per rectum, which was performed 60 to 130 d following AI. Pregnancy rate data were analyzed by Chi-square. RIWJLTS AND DISCUSSION Experiment 1 Documentation of proper semen placement was an important consideration in this study. Semen was assumed to be deposited into the body of the uterus, but recent research has indicated that only 40% of all inseminations are actually in the uterine body (13). Therefore, right vs left side effects on numbers of sperm cells recovered were important and are presented below (Table 1). There was no significant effect of side (PmO.61) on numbers of sperm cells recovered. Data were consistent for each segment of the oviduct, except for the upper third segment, where a disproportion occurred. However, the standard errors were large, indicating the existence of large variation. Also, the side effect did not significantly interact with any of the other main effects. Thus, semen placement in this study was considered acceptable. Supporting this assumption is the demonstration that intracornual semen deposition that is no deeper than 5 cm apparently does not affect sperm cell migration to the contralataral horn and therefore should not affect pregnancy rates (14).
Table 1. Right vs left side effects on numbers of sperm cells recovered from the oviducts of beef cows 16 h after artificial inseminationa
Side of oviduct Right Left
No. of sperm cells recovered per oviduct Lower Middle Upper third third third Totalb 353 371'
32 18
4 164
389 553
118 203 L 16 219 Average SEM Cows were inseminated with 100 x 10' sperm cells 10 to 12 h after icing observed in estrus. The number of sperm cells recovered was not affected by side of the oviduct (P-0.61). The side by segment interaction was not significant.
While the actual number of sperm cells recovered 16 h after AI was higher for PGF-treated cows (Table 2), the difference was not
112
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
significant (P=O.71). The large standard error indicates the high amount of variation observed among cows within treatment. Also, sperm cells were not recovered from all segments of all cows. There was one control and one treated cow from which no sperm cells were recovered at all. These results are contrary to those observed in rabbits (3,7) and in sheep (5), which showed as much as a ten-fold increase in numbers of sperm cells in the oviducts for PGF-treated animals compared with saline-treated animals post insemination. However, an increase in sperm numbers in the oviducts of ewes receiving various doses of PGF was not observed in one experiment (15).
Table 2. The effect of 25 mg of prostaglandin Fzu on the number of sperm cells recovered from various segments of the oviducts of beef cows 16 h after artificial insemination
Treatment Prostaglandin Control Overall
No. of cows 7 7 14
No. of sperm cells recovered per oviduct Middle Lower Upper third third Total third 464(3>b 261il) 363
14(5) 3664) 25
164(6) 366) 84
642' 3ooc 472
Average SEM 140 L 11 82 231 "Cows were inseminated with 100 x 10" sperm cells 10 to 12 h after eing observed in estrus. Numbers in parentheses are the number of cows in which no sperm $; 1s were recovered from that segment of the oviduct. +Jeans in the same row or column with different superscript letters are different (P
B
The total number of sperm cells recovered per animal in our study is similar to that of previous reports (5.7). Twelve hundred sperm cells were observed in the oviducts of control rabbits 3 b after mating but there were 7,000 in PGF-treated does (7). This observation occurred with an inseminate containing 102 million sperm cells, which was similar to the number used in our study. Likewise, 1,300 to 1,400 sperm cells were recovgred from sheep oviducts 24 h following an insemination of 300 x 10 cells (5). An increase in sperm cell numbers in the oviducts has been observed up to 8 h in cattle following uterine insemination with fresh semeg but declined thereafter (8,9). In these studies, where 2,000 x 10 cells were used in the inseminate, 17,000 and 15,000 sperm cells were recovered at 16 and 24 h post insemination. Counts of sperm cells in tract sections (16) revealed a progressive increase in the numbers of sperm'cells present in the oviducts during a 2- to 18-h period. Thus, sufficient sperm cells are apparently not established in the oviducts until 8 to 12 h or longer after mating.
JULY 1988 VOL. 30 NO. 1
113
THERIOGENOLOGY
A possible explanation for the failure to obtain increased of sperm cell numbers in the oviducts may have been the dose _ level _ PGF. A 0.75-mg dose of PGF to rabbit does was reported to be near optimal for increasing sperm cell numbers in the reproductive tract (3). Since this was equal to 0.1875 mg/kg of body weight, it would be equivalent to an 84-mg dose in a 450-kg cow. However, positive results were obtained in ewes with a 2.5-mg dose (5). This dose would be approximately equal to a 17-mg dose in a 450-kg cow. The 25-mg dose used in our experiment was selected because it is known to be effective in causing other physiological events in beef cattle (17). The number of sperm cells recovered from the oviducts was significantly higher in the lower third segment which included the utero-tubal junction than in the middle or upper third segments. Approximately 75% of the sperm cells recovered from each oviduct were located in the lower third segment (isthmus and utero-tubal junction) for both PGF and control cows (Table 2). It has been suggested that there is a preovulatory accumulation and sequestering of sperm cells in the lower part of the oviducts with an active phase of release near the time of ovulation in rabbits, hamsters and pigs (17). Also, approximately 8 h is required to establish this pool of sperm cells in the isthmus of sheep. This would substantiate the observation that the highest number of sperm cells in the oviducts of cattle occurred 8 h following insemination (8). The folds of the isthmus and utero-tubal junction of cattle have been documented (16) as a storage point for sperm cells that are protected there from phagocytosis. These researchers also reported that very few sperm ever reach the upper tube (ampulla). Approximately 30,000 sperm were recovered (19) from the utero-tubal junction-isthmus area of the genital tract of cows slaughtered 12 h after a uterine insemination of one billion cells. No sperm were recovered from the ampullae. Likewise, in our study, sperm cells were recovered from the upper oviduct of only one cow in each treatment group. The effect of ovulation on the number of sperm cells recovered from the oviducts is presented in Table 3. There were 7114 cows that had ovulated at the time of slaughter, and there was a tendency (P=O.lO) for more sperm cells to be recovered from these cows than from preovulatory cows. There was no ovulation by side interaction, indicating that there were just as many sperm recovered from the contralateral as the ipsilateral side of the ovulation within cow. Five of the seven cows that had ovulated by the time of slaughter had been treated with PGF at insemination. However, the treatment by ovulation interaction was not significant. There is evidence that sperm cells move from the lower to the upper oviduct at the time of ovulation in sheep (18). A trend toward adovarian movement of sperm cells in the oviduct has been observed in cattle, although the number of observations is small (20). An additional objective of our study had been to determine the number of abnormal vs normal sperm cells reaching the oviducts of cattle. The semen selected for insemination had a moderate level of
114
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
primary (head) abnormalities but a high level of secondary (mid-piece) and tertiary (tail) abnormalities (12%). Unfortunately, too few sperm cells were recovered in our study to allow meaningful results to be reported for normal vs abnormal sperm cells reaching the oviduct. Retrograde movement and massive loss of sperm cells following'uterine insemination in virgin heifers has recently been reported (21). These findings were corroborated (19) with the observation that 92% of the sperm cells inseminated into the uterus had displaced into the vagina. Further, proportions of both tertiary and total abnormal sperm cells were lower in the uter-tubal junction-isthmus than in either the inseminate or the uterus. This suggests that there is differential retention of abnormal sperm cells within the bovine genital tract. This fact, coupled with the probable retrograde sperm cell losses , could have affected the numbers of sperm cells reaching the oviduct in our study.
Table 3. The effect of ovulation on number of sperm cells recovered from various segments of the oviducts of beef cows 16 h after artificial insemination
Ovulation status Preovulatory Ovulated
No. of cows 7 7
No. of sperm cells recovered per oviduct Middle Lower Upper third third third Total 171 (2) 553 (2)
25 (4) 25 (5)
4 (6) 164 (6)
200 742
< 16 124 231 Average SEM 214 *Cows were inseminated with 100 x 10' sperm cells 10 to 12 h after Being observed in estrus. The number of sperm cells recovered tended to be greater in those gows that had ovulated at the time of slaughter (P=O.lO). Numbers in parentheses are the number of cows from which no sperm cells were recovered from that segment of the oviduct. Experiment 2 Direct application of the hypothesis that PGF might increase the number of sperm cells reaching the sight of ovulation following AI in cattle (Experiment 1) was studied by determining pregnancy rates of beef cattle females treated i.m. with either PGF or vehicle immediately after AI in our experiment. Since higher ovum fertilization rates had been obtained in rabbit does following insemination accompanied by administration of PGF (4), perhaps higher pregnancy rates could b& obtained in cattle using a similar procedure. The use of 20 x 10 sperm cells per semen unit in our experiment is the usual semen unit size for AI in beef cattle. There was no significant effect of PGF on pregnancy rates in either crossbred heifers or in postpartum suckled cows (Table 4). The pooled pregnancy rates of treated and control females for both
JULY 1988 VOL. 30 NO. 1
115
THERIOGENOLOGY
classes of animals were identical (56.1%). These were first service inseminations only. While differences were not statistically significant, the direction of the response between the heifers and the lactating cows was opposite. It is not known whether this is a breed type, parity or technician effect since all of these factors were totally confounded with location. Increased pregnancy rates in cows, but not in yearling heifers, has been obtained after a lo-set clitoral massage which followed AI (22). The hypothesis was that mating stimuli resulted in an oxytocin release from the pituitary, which may have affected sperm cell transport to the site of fertilization (23). However, increased pregnancy rates were not obtained following injection of oxytocin at AI (24). Further, there was no difference in pregnancy rates between yearling heifers and parous cows receiving oxytocin, which were 49.5 and 58.4% for treated and untreated females, respectively.
Table 4.
Pregnancy rates of beef females after administration of immediately following artificial insemination at
Prostaglandin No. bred pregnant % NO.
Breed type
No. bred
Control No. pregnant
%
70.6 34 24 34 20 58.8 34.8 23 8 23 12 52.2 56.1 57 32 57 32 56.1 L eFemales were AI with 20 x 10' sperm cells 10 to 12 h after being gbserved in estrus. There was no significant difference in pregnancy rate between treatment groups for either breed type. Angus x Hereford heifers Chianina-cross cows Total
In summary, these data support the hypothesis that there is preovulatory accumulation and possible sequestering of spermatozoa in the caudal isthmus of the oviducts of cattle after AI. That this could be the functional sperm reservoir for ovulation seems plausible. These data also suggest that a 25-mg dose of PGF administered i.m. to beef cattle following AI does not affect the number of sperm cells present in the oviduct at the approximate time of ovulation and therefore does not affect the pregnancy rate. However, there was a discrepancy in the effect of PGF on pregnancy rate between yearling heifers and parous cows, with a nonsignificant tendency toward improvement in the latter group. Perhaps a larger dose used on a larger sample of parous cows would prove more effective.
116
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
REFERENCES 1.
Bellows, R.A., Short, R.E. and Staigmiller, R.B. Research areas in beef cattle reproduction. In: Hawk, H.W. (ed.). Beltsville Symposia in Agricultural Research III. Animal Reproduction. Allanheld, Osmun, Montclair, 1979, p. 3.
2.
Hawk, H.W. Infertility in dairy cattle. In: Hawk, H.W., (ea.). Beltsville Symposia in AgriculturarResearch III. Animal Reproduction. Allanheld, Osmun, Montclair, 1979, p. 19.
3.
Hawk, H.W., Cooper, B.S. and Conley, H.H. Effect of acetylcholine, prostaglandin F,ccand estradiol on number of sperm in the reproductive tract of inseminated rabbits. J. Anim. Sci. -55:891-900 (1982).
4.
Spilman, C.H., Finn, A.E. and Norland, J.F. Effect of prostaglandin on sperm transport and fertilization in the rabbit. Prostaglandins 4:57-60 (1973).
5.
Edquist, S., Einarsson, S. and Gustafsson, B. Effect of prostaglandin F,o on sperm transport in the reproductive tract of the ewe. Acta Vet. Stand. -16:149-156 (1975).
6.
Gustafsson, B., Graham, E.F., Crabo, B.G., Pavelko, M.K. and Wagner, W.C. Prefreeze supplementation of ram semen with PGE, and PGFzcr:effects of sperm vitality in vitro and on sperm transport in the ewe. Proc. Ann. Meet. Sot. Study Reprod., p, 70 (1977).
7.
Hawk, H.W. and Cooper, B.S. Increased retention of spermatozoa in the reproductive tract of estrous rabbits after administration of prostaglandin Fza immediately before insemination. J. Anim. Sci. -49:154-157 (1979).
8.
El Banna, A.A. and Hafez, E.S.E. Sperm transport and distribution in rabbit and cattle female tract. Fertil. Steril. -21:534-541 (1970).
9.
Dobrowolski, D. and Hafez, E.S.E. Transport and distribution of spermatozoa in the reproductive tract of the cow. J. Anim. sci. -31:940-943 (1970).
10.
Chandler, J.E., Atkinson, R.W., Hay, G.M. and Crain, R.L. Environmental and genetic sources of variation for semen quality in mature Holstein bulls. J. Dairy Sci. -68:1270-1279 (1985).
11.
Johnson, L., Berndtson, W.E. and Pickett, B.W. An improved method for evaluating acrosomes of bovine spermatozoa. J. Anti. Sci. 42:951-954 (1976).
12.
SAS Institute Inc. SAS/STAT Guide for Personal Computers. Gary, UC, SAS Institute Inc., 1985, pp. 183-259.
JULY 1988 VOL. 30 NO. I
117
THERIOGENOLOGY
13.
Peters, J.L., Senger, P.L., Rosenberger, J.L. and O'Conner, M.L. Radiographic evaluation of bovine artificial inseminating technique among professional and herdsman-inseminators using .5- and .25-ml french straws. J. Anim. Sci. -59:167f-1683 (1984).
14.
Fernandez-Van Cleve, J., Zavos, P.M., Salim, B. and Hemken, R.W. Effect of intracornual depth of semen deposition on conception rate and embryonic retardation in superovulated cows. Kentucky Ag. Exp. Sta. 98th Ann. Rep., pp. 156-157 (1985).
15.
Hawk, H.W. and Cooper, B.S. Improvement by ergonovine of sperm transport, fertilization and pregnancy rates in ewes in natural or prostaglandin-induced estrus. J. Anim. Sci. -59:754-763 (1984).
16.
Thibault, C., Gerard, M. and Heyman, Y. Transport, survival and fertilizing ability of vertebrate spermatozoa. INSERM, Paris, pp. 343-356 (1973).
17.
Inskeep, E.K. Potential uses of prostaglandins in control of reproductive cycles of domestic animals. J. Anim. Sci. -36:1149-1157 (1973).
18.
Hunter, R.H.F., Barwise, L. and King, R. Sperm transport, storage and release in the sheep oviduct in relation to the time of ovulation. Brit. Vet. J. -138:225-232 (1982).
19.
Mitchell, J.R., Senger, P.L. and Rosenberger, J.L. Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract. J. Anim. Sci. -61:956-967 (1985).
20.
Hunter, R.H.F. and Wilmut, I. Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod. Nutr. Develop. %:597-608 (1984).
21.
Larsson, B. Sperm distribution in artificially inseminated heifers, a preliminary report. 10th Int. Congr. Reprod. Artif. Insemin., Champaign-Urbana, 3:372-374 (1984).
22.
Randel, R.D., Short, R.E., Christenson, D.S. and Bellows, R.A. Effect of clitoral massage after artificial insemination on conception in the bovine. J. Anim. Sci. -40:1119-1123 (1975).
23.
Hays, R.L. and Van DeMark, N.L. Effect of stimulation of the reproductive organs of the cow on the release of an oxytocin-like substance. Endocrinol. -52:634-637 (1953).
24.
Morrison, D.G., Humes, P.E., Peterson, F.J. and Godke, R.A. Oxytocin following artificial insemination in crossbred beef cattle. J. Anim. Sci. -45(Suppl. 1):421 (1977).
118
JULY 1988 VOL. 30 NO. 1
EFFECT OF PROSTAGLANDIN Flu AT INSEMINATION ON SPERM CELL NUMBERS AND PREGNANCY RATE IN BEEF CATTLE D. G. Morrison, 1,4 J. E2 Chandler,' L. S.3Chandler,* A. F. Radintz and M. B. Warner 1Rosepine Research Station and *Department of Dairy Science Louisiana State University Agricultural Center Baton Rouge, LA 70803 Received for publication: July 22, 1987 Accepted: March 22, 1988 ABSTRACT Fourteen cycling, nonlactating, multiparous beef cows were artificially inseminated (AI) 10 to 12 h after the onset of atural % estrus. One unit of frozen-thawed semen containing 100 x 10 total sperm cells was deposited into the body of the uterus. Immediately after AI, alternating cows were injected i.m. with either 25 mg (5 ml) of prostaglandin F20.(PGF)or 5 ml of 0.9% saline-benzyl alcohol control solution. Cows were slaughtered 16 + 1 h post AI, oviducts were retrieved, segmented into thirds (upper, middle and lower) and flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline. The number of sperm cells was counted using a phase contrast microscope. There were no right or left side effects (P=O.61) on the number of sperm cells recovered per oviduct within cow (389 vs 553; average SEM = 219). PGF had no effect (P=O.77) on the number of sperm cells recovered per oviduct (642 vs 300; average SEM = 231 for PGF and control females, respectively). More sperm cells were recovered from the lower third segment (P
prostaglandin, artificial insemination, sperm cells. pregnancy, beef cattle Acknowledgments 3 Former Associate, Idlewild Research Station, Clinton, LA 70722. 4 Reprint requests. Rosepine Research Station, P.O. Box 26, Rosepine, LA 70659. Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 87-92-1387.
JULY 1988 VOL. 30 NO. 1
109
THERIOGENOLOGY
INTRODUCTION Experimental treatments that improve sperm cell transport in laboratory animals may have relevance to problems of fertilization failure in larger animals. Fertilization failure in cattle is almost as.detrimental as embryonic mortality in reducing or delaying pregnancy (1,2). Prostaglandin F20(PGF) administration to rabbit does near the time of insemination has been reported to significantly increase the number of sperm cells in the oviducts 2.5 h after insemination (3). Higher ovum fertilization rates were also obtained when PGF was injected into does (4). When PGF was added to the inseminate or injected at the time of insemination of ewes, more sperm cells were recovered from the oviducts of treated than untreated ewes 16 or 24 h after insemination (5,6). PGF has been shown to not only increase contractility of the reproductive tract, thus increasing sperm cell transport, but also to reduce the normal loss of sperm cells from the reproductive tract, which is normally due to drainage or expulsion to the exterior. Thus more cells are available for transport to the upper tract (7). The effect of administration of PGF on sperm cell number and pregnancy rate has not beep assessed in cattle. The number of sperm cells in bovine oviducts has been shown to be highest between 8 and 16 h after insemination (8,9). The widespread use of AI in beef and dairy cattle makes the possibility of using an injectable compound to decrease services per pregnancy attractive. The objective of our investigation was to determine if PGF injection at the time of AI would increase the number of sperm cells .in the oviducts of cattle at about the time of ovulation and therefore increase the pregnancy rate.
MATERIALS AND METHODS Experiment 1 Fourteen Angus and Angus x Hereford, nonlactating, multiparous beef cows were used in this portion of the study. Ages ranged from 3 to 11 yr, and body condition was average to above average. All females had weaned at least one calf and were therefore considered to be fertile animals. Cows were observed for estrous activity during two consecutive estrous cycles to determine if cycles were typical in length (18 to 23 d) and if observable standing estrus was occurring. A surgically altered teaser bull wearing a chin-ball marking harness was kept with the cows as an aid in detecting estrus. Semen was collected fro% a mature'Jersey bull and packaged for freezing to contain 100 x 10 total sperm cells per 0.5 ml straw. Post thaw semen evaluation was performed as previously described (IO). Two units of semen were selected at random from each batch
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
for evaluation. Progressive motility, measured at 0 h post thaw and after 3 h of incubation at 37°C was 35 and 25X, respectively. Percentage of intact acrosomes and percentage of primary and secondary abnormalities were 65, 30 and 12%. respectively. Cows were artificially inseminated with a single unit of semen 10 to 12 h after being observed in estrus. Estrus observations were made at 0700 and 1800 h each day. Semen straws were thawed in a warm water bath (38'C) for 20 sec. Semen was deposited at the rim of the internal OS cervix and was therefore assumed to be in the body of the uterus. Immediately after AI (maximum of 1 min), alternate cows were injected i.m. with either 25 mg (5 ml) of PGF (Lutalyse, dinoprost tromethamine; The Upjohn Co., Kalamazoo, MI) or 5 ml of 0.9% saline-bensyl alcohol control solution. Females were transported approximately 12 km to a local slaughter facility and slaughtered 16 5 1 h after AI. As soon as possible (30 to 45 min), the reproductive tract was retrieved and the oviducts were ligated and excised. Examination of the ovaries revealed either a large (15 to 21 mm) follicle or an ovulation stigma for each cow. Oviducts were brought back to the laboratory where each oviduct (uterine tube) was sectioned into three equal The lower segment also segments (upper, middle and lower). included the utero-tubal junction and approximately 1 mm of the tip of the uterine horn. Each segment was flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline by the same technician (11). A minimum of two, lo-u1 smears of each flushing sample was counted. For each smear, the entire surface was scanned using a phase contrgst microscope (160X) to insure a count per 10 ul. Use of 100 x 10 sperm cells per insemination unit also helped to insure that sperm cells were available for6counting since this is five times the normal AI unit of 20 x 10 cells. Data were analyzed by a general linear models procedure (12). The model included the effects of treatment, side, ovulation, segment and all first order interactions. Treatment, side and segment were considered fixed effects, while ovulation was considered random. Cow within treatment by ovulation was used as the error term for testing treatment, ovulation and treatment by ovulation. Residual error was used for the other main effects and interactions. Experiment 2 One hundred and fourteen beef females (68 Angus x Hereford heifers and 46 Chianina crossbred postpartum suckled cows) were used to determine if PGF administered at AI would affect pregnancy rate. The two sets of females were maintained at different locations and inseminated by a single technician at each location. Surgically altered teaser bulls equipped with chin-ball marking devices were kept with the herd at all times as an aid in estrus detection. Cows were visually observed at least twice daily (0700 and 1800 h) for signs of estrus. Thgse observed in estrus were inseminated 10 to 12 h later with 20 x 10 motile sperm cells in 0.5~ml straws. Frozen
JULY 1988 VOL. 30 NO.’ 1
111
I’HERIOGENOLOGY
semen from a single Angus bull was used to inseminate the Angus x Hereford heifers. Semen from 16 Chianina sires was used at random to inseminate the Chianina cows. Semen was thawed as described in Experiment 1. Immediately after AI (maximum of 1 min), alternating females were administered PGF or vehicle i.m. as described above. Pregnancy'rates were determined by palpation per rectum, which was performed 60 to 130 d following AI. Pregnancy rate data were analyzed by Chi-square. RIWJLTS AND DISCUSSION Experiment 1 Documentation of proper semen placement was an important consideration in this study. Semen was assumed to be deposited into the body of the uterus, but recent research has indicated that only 40% of all inseminations are actually in the uterine body (13). Therefore, right vs left side effects on numbers of sperm cells recovered were important and are presented below (Table 1). There was no significant effect of side (PmO.61) on numbers of sperm cells recovered. Data were consistent for each segment of the oviduct, except for the upper third segment, where a disproportion occurred. However, the standard errors were large, indicating the existence of large variation. Also, the side effect did not significantly interact with any of the other main effects. Thus, semen placement in this study was considered acceptable. Supporting this assumption is the demonstration that intracornual semen deposition that is no deeper than 5 cm apparently does not affect sperm cell migration to the contralataral horn and therefore should not affect pregnancy rates (14).
Table 1. Right vs left side effects on numbers of sperm cells recovered from the oviducts of beef cows 16 h after artificial inseminationa
Side of oviduct Right Left
No. of sperm cells recovered per oviduct Lower Middle Upper third third third Totalb 353 371'
32 18
4 164
389 553
118 203 L 16 219 Average SEM Cows were inseminated with 100 x 10' sperm cells 10 to 12 h after icing observed in estrus. The number of sperm cells recovered was not affected by side of the oviduct (P-0.61). The side by segment interaction was not significant.
While the actual number of sperm cells recovered 16 h after AI was higher for PGF-treated cows (Table 2), the difference was not
112
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
significant (P=O.71). The large standard error indicates the high amount of variation observed among cows within treatment. Also, sperm cells were not recovered from all segments of all cows. There was one control and one treated cow from which no sperm cells were recovered at all. These results are contrary to those observed in rabbits (3,7) and in sheep (5), which showed as much as a ten-fold increase in numbers of sperm cells in the oviducts for PGF-treated animals compared with saline-treated animals post insemination. However, an increase in sperm numbers in the oviducts of ewes receiving various doses of PGF was not observed in one experiment (15).
Table 2. The effect of 25 mg of prostaglandin Fzu on the number of sperm cells recovered from various segments of the oviducts of beef cows 16 h after artificial insemination
Treatment Prostaglandin Control Overall
No. of cows 7 7 14
No. of sperm cells recovered per oviduct Middle Lower Upper third third Total third 464(3>b 261il) 363
14(5) 3664) 25
164(6) 366) 84
642' 3ooc 472
Average SEM 140 L 11 82 231 "Cows were inseminated with 100 x 10" sperm cells 10 to 12 h after eing observed in estrus. Numbers in parentheses are the number of cows in which no sperm $; 1s were recovered from that segment of the oviduct. +Jeans in the same row or column with different superscript letters are different (P
B
The total number of sperm cells recovered per animal in our study is similar to that of previous reports (5.7). Twelve hundred sperm cells were observed in the oviducts of control rabbits 3 b after mating but there were 7,000 in PGF-treated does (7). This observation occurred with an inseminate containing 102 million sperm cells, which was similar to the number used in our study. Likewise, 1,300 to 1,400 sperm cells were recovgred from sheep oviducts 24 h following an insemination of 300 x 10 cells (5). An increase in sperm cell numbers in the oviducts has been observed up to 8 h in cattle following uterine insemination with fresh semeg but declined thereafter (8,9). In these studies, where 2,000 x 10 cells were used in the inseminate, 17,000 and 15,000 sperm cells were recovered at 16 and 24 h post insemination. Counts of sperm cells in tract sections (16) revealed a progressive increase in the numbers of sperm'cells present in the oviducts during a 2- to 18-h period. Thus, sufficient sperm cells are apparently not established in the oviducts until 8 to 12 h or longer after mating.
JULY 1988 VOL. 30 NO. 1
113
THERIOGENOLOGY
A possible explanation for the failure to obtain increased of sperm cell numbers in the oviducts may have been the dose _ level _ PGF. A 0.75-mg dose of PGF to rabbit does was reported to be near optimal for increasing sperm cell numbers in the reproductive tract (3). Since this was equal to 0.1875 mg/kg of body weight, it would be equivalent to an 84-mg dose in a 450-kg cow. However, positive results were obtained in ewes with a 2.5-mg dose (5). This dose would be approximately equal to a 17-mg dose in a 450-kg cow. The 25-mg dose used in our experiment was selected because it is known to be effective in causing other physiological events in beef cattle (17). The number of sperm cells recovered from the oviducts was significantly higher in the lower third segment which included the utero-tubal junction than in the middle or upper third segments. Approximately 75% of the sperm cells recovered from each oviduct were located in the lower third segment (isthmus and utero-tubal junction) for both PGF and control cows (Table 2). It has been suggested that there is a preovulatory accumulation and sequestering of sperm cells in the lower part of the oviducts with an active phase of release near the time of ovulation in rabbits, hamsters and pigs (17). Also, approximately 8 h is required to establish this pool of sperm cells in the isthmus of sheep. This would substantiate the observation that the highest number of sperm cells in the oviducts of cattle occurred 8 h following insemination (8). The folds of the isthmus and utero-tubal junction of cattle have been documented (16) as a storage point for sperm cells that are protected there from phagocytosis. These researchers also reported that very few sperm ever reach the upper tube (ampulla). Approximately 30,000 sperm were recovered (19) from the utero-tubal junction-isthmus area of the genital tract of cows slaughtered 12 h after a uterine insemination of one billion cells. No sperm were recovered from the ampullae. Likewise, in our study, sperm cells were recovered from the upper oviduct of only one cow in each treatment group. The effect of ovulation on the number of sperm cells recovered from the oviducts is presented in Table 3. There were 7114 cows that had ovulated at the time of slaughter, and there was a tendency (P=O.lO) for more sperm cells to be recovered from these cows than from preovulatory cows. There was no ovulation by side interaction, indicating that there were just as many sperm recovered from the contralateral as the ipsilateral side of the ovulation within cow. Five of the seven cows that had ovulated by the time of slaughter had been treated with PGF at insemination. However, the treatment by ovulation interaction was not significant. There is evidence that sperm cells move from the lower to the upper oviduct at the time of ovulation in sheep (18). A trend toward adovarian movement of sperm cells in the oviduct has been observed in cattle, although the number of observations is small (20). An additional objective of our study had been to determine the number of abnormal vs normal sperm cells reaching the oviducts of cattle. The semen selected for insemination had a moderate level of
114
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
primary (head) abnormalities but a high level of secondary (mid-piece) and tertiary (tail) abnormalities (12%). Unfortunately, too few sperm cells were recovered in our study to allow meaningful results to be reported for normal vs abnormal sperm cells reaching the oviduct. Retrograde movement and massive loss of sperm cells following'uterine insemination in virgin heifers has recently been reported (21). These findings were corroborated (19) with the observation that 92% of the sperm cells inseminated into the uterus had displaced into the vagina. Further, proportions of both tertiary and total abnormal sperm cells were lower in the uter-tubal junction-isthmus than in either the inseminate or the uterus. This suggests that there is differential retention of abnormal sperm cells within the bovine genital tract. This fact, coupled with the probable retrograde sperm cell losses , could have affected the numbers of sperm cells reaching the oviduct in our study.
Table 3. The effect of ovulation on number of sperm cells recovered from various segments of the oviducts of beef cows 16 h after artificial insemination
Ovulation status Preovulatory Ovulated
No. of cows 7 7
No. of sperm cells recovered per oviduct Middle Lower Upper third third third Total 171 (2) 553 (2)
25 (4) 25 (5)
4 (6) 164 (6)
200 742
< 16 124 231 Average SEM 214 *Cows were inseminated with 100 x 10' sperm cells 10 to 12 h after Being observed in estrus. The number of sperm cells recovered tended to be greater in those gows that had ovulated at the time of slaughter (P=O.lO). Numbers in parentheses are the number of cows from which no sperm cells were recovered from that segment of the oviduct. Experiment 2 Direct application of the hypothesis that PGF might increase the number of sperm cells reaching the sight of ovulation following AI in cattle (Experiment 1) was studied by determining pregnancy rates of beef cattle females treated i.m. with either PGF or vehicle immediately after AI in our experiment. Since higher ovum fertilization rates had been obtained in rabbit does following insemination accompanied by administration of PGF (4), perhaps higher pregnancy rates could b& obtained in cattle using a similar procedure. The use of 20 x 10 sperm cells per semen unit in our experiment is the usual semen unit size for AI in beef cattle. There was no significant effect of PGF on pregnancy rates in either crossbred heifers or in postpartum suckled cows (Table 4). The pooled pregnancy rates of treated and control females for both
JULY 1988 VOL. 30 NO. 1
115
THERIOGENOLOGY
classes of animals were identical (56.1%). These were first service inseminations only. While differences were not statistically significant, the direction of the response between the heifers and the lactating cows was opposite. It is not known whether this is a breed type, parity or technician effect since all of these factors were totally confounded with location. Increased pregnancy rates in cows, but not in yearling heifers, has been obtained after a lo-set clitoral massage which followed AI (22). The hypothesis was that mating stimuli resulted in an oxytocin release from the pituitary, which may have affected sperm cell transport to the site of fertilization (23). However, increased pregnancy rates were not obtained following injection of oxytocin at AI (24). Further, there was no difference in pregnancy rates between yearling heifers and parous cows receiving oxytocin, which were 49.5 and 58.4% for treated and untreated females, respectively.
Table 4.
Pregnancy rates of beef females after administration of immediately following artificial insemination at
Prostaglandin No. bred pregnant % NO.
Breed type
No. bred
Control No. pregnant
%
70.6 34 24 34 20 58.8 34.8 23 8 23 12 52.2 56.1 57 32 57 32 56.1 L eFemales were AI with 20 x 10' sperm cells 10 to 12 h after being gbserved in estrus. There was no significant difference in pregnancy rate between treatment groups for either breed type. Angus x Hereford heifers Chianina-cross cows Total
In summary, these data support the hypothesis that there is preovulatory accumulation and possible sequestering of spermatozoa in the caudal isthmus of the oviducts of cattle after AI. That this could be the functional sperm reservoir for ovulation seems plausible. These data also suggest that a 25-mg dose of PGF administered i.m. to beef cattle following AI does not affect the number of sperm cells present in the oviduct at the approximate time of ovulation and therefore does not affect the pregnancy rate. However, there was a discrepancy in the effect of PGF on pregnancy rate between yearling heifers and parous cows, with a nonsignificant tendency toward improvement in the latter group. Perhaps a larger dose used on a larger sample of parous cows would prove more effective.
116
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
REFERENCES 1.
Bellows, R.A., Short, R.E. and Staigmiller, R.B. Research areas in beef cattle reproduction. In: Hawk, H.W. (ed.). Beltsville Symposia in Agricultural Research III. Animal Reproduction. Allanheld, Osmun, Montclair, 1979, p. 3.
2.
Hawk, H.W. Infertility in dairy cattle. In: Hawk, H.W., (ea.). Beltsville Symposia in AgriculturarResearch III. Animal Reproduction. Allanheld, Osmun, Montclair, 1979, p. 19.
3.
Hawk, H.W., Cooper, B.S. and Conley, H.H. Effect of acetylcholine, prostaglandin F,ccand estradiol on number of sperm in the reproductive tract of inseminated rabbits. J. Anim. Sci. -55:891-900 (1982).
4.
Spilman, C.H., Finn, A.E. and Norland, J.F. Effect of prostaglandin on sperm transport and fertilization in the rabbit. Prostaglandins 4:57-60 (1973).
5.
Edquist, S., Einarsson, S. and Gustafsson, B. Effect of prostaglandin F,o on sperm transport in the reproductive tract of the ewe. Acta Vet. Stand. -16:149-156 (1975).
6.
Gustafsson, B., Graham, E.F., Crabo, B.G., Pavelko, M.K. and Wagner, W.C. Prefreeze supplementation of ram semen with PGE, and PGFzcr:effects of sperm vitality in vitro and on sperm transport in the ewe. Proc. Ann. Meet. Sot. Study Reprod., p, 70 (1977).
7.
Hawk, H.W. and Cooper, B.S. Increased retention of spermatozoa in the reproductive tract of estrous rabbits after administration of prostaglandin Fza immediately before insemination. J. Anim. Sci. -49:154-157 (1979).
8.
El Banna, A.A. and Hafez, E.S.E. Sperm transport and distribution in rabbit and cattle female tract. Fertil. Steril. -21:534-541 (1970).
9.
Dobrowolski, D. and Hafez, E.S.E. Transport and distribution of spermatozoa in the reproductive tract of the cow. J. Anim. sci. -31:940-943 (1970).
10.
Chandler, J.E., Atkinson, R.W., Hay, G.M. and Crain, R.L. Environmental and genetic sources of variation for semen quality in mature Holstein bulls. J. Dairy Sci. -68:1270-1279 (1985).
11.
Johnson, L., Berndtson, W.E. and Pickett, B.W. An improved method for evaluating acrosomes of bovine spermatozoa. J. Anti. Sci. 42:951-954 (1976).
12.
SAS Institute Inc. SAS/STAT Guide for Personal Computers. Gary, UC, SAS Institute Inc., 1985, pp. 183-259.
JULY 1988 VOL. 30 NO. I
117
THERIOGENOLOGY
13.
Peters, J.L., Senger, P.L., Rosenberger, J.L. and O'Conner, M.L. Radiographic evaluation of bovine artificial inseminating technique among professional and herdsman-inseminators using .5- and .25-ml french straws. J. Anim. Sci. -59:167f-1683 (1984).
14.
Fernandez-Van Cleve, J., Zavos, P.M., Salim, B. and Hemken, R.W. Effect of intracornual depth of semen deposition on conception rate and embryonic retardation in superovulated cows. Kentucky Ag. Exp. Sta. 98th Ann. Rep., pp. 156-157 (1985).
15.
Hawk, H.W. and Cooper, B.S. Improvement by ergonovine of sperm transport, fertilization and pregnancy rates in ewes in natural or prostaglandin-induced estrus. J. Anim. Sci. -59:754-763 (1984).
16.
Thibault, C., Gerard, M. and Heyman, Y. Transport, survival and fertilizing ability of vertebrate spermatozoa. INSERM, Paris, pp. 343-356 (1973).
17.
Inskeep, E.K. Potential uses of prostaglandins in control of reproductive cycles of domestic animals. J. Anim. Sci. -36:1149-1157 (1973).
18.
Hunter, R.H.F., Barwise, L. and King, R. Sperm transport, storage and release in the sheep oviduct in relation to the time of ovulation. Brit. Vet. J. -138:225-232 (1982).
19.
Mitchell, J.R., Senger, P.L. and Rosenberger, J.L. Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract. J. Anim. Sci. -61:956-967 (1985).
20.
Hunter, R.H.F. and Wilmut, I. Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod. Nutr. Develop. %:597-608 (1984).
21.
Larsson, B. Sperm distribution in artificially inseminated heifers, a preliminary report. 10th Int. Congr. Reprod. Artif. Insemin., Champaign-Urbana, 3:372-374 (1984).
22.
Randel, R.D., Short, R.E., Christenson, D.S. and Bellows, R.A. Effect of clitoral massage after artificial insemination on conception in the bovine. J. Anim. Sci. -40:1119-1123 (1975).
23.
Hays, R.L. and Van DeMark, N.L. Effect of stimulation of the reproductive organs of the cow on the release of an oxytocin-like substance. Endocrinol. -52:634-637 (1953).
24.
Morrison, D.G., Humes, P.E., Peterson, F.J. and Godke, R.A. Oxytocin following artificial insemination in crossbred beef cattle. J. Anim. Sci. -45(Suppl. 1):421 (1977).
118
JULY 1988 VOL. 30 NO. 1