Comparing early embryo mortality in dairy cows during hot and cool seasons of the year

Theriogenology 39:7 19-737, 1993

COMPARING

EARLY EMBRYO MORTALITY IN DAIRY COWS DURING HOT AND COOL SEASONS OF THE YEAR

D.P. Ryan,‘V2VaJ.F. Prichard, I E. Kopel’ and R.A. Godkeipb ‘Animal Science Department, LAES, LSU Agricultural Center, Louisiana State University, Baton Rouge, LA 70803 USA and *Masstock Research & Development Corporation Riyadh 11492, Saudi Arabia Received for publication: Accepted:

A~pst June

I, 27,

1991 1992

ABSTRACT The objectives of this study were to identify the level and stage of embryonic mortality that occur in dairy cows during hot and cool seasons of the year. Experimental dairy cows, of varying ages, were artificially inseminated with frozen-thawed semen from proven Holstein sires. Females on each dairy unit were then randomly allocated to one of three experimental groups after partitioning by day of artificial insemination, days post partum, parity, and current milk production level. In Group I and Group II, nonsurgical embryo collection was performed on each cow using Dulbecco’s phosphate-buffered saline as the flushing medium. Embryos from cows in Group I were collected on Days 6 or 7 post insemination during the hot (n =93) and cool (n =64) seasons. Embryos from cows in Group II were collected on Days 13 or 14 post insemination during the hot (n=97) and cool (n=63) seasons. In Group III, contemporary control cows were also inseminated during the hot (n= 106) and cool (n=106) seasons, and fetal heart beat was Embryo evaluated via ultrasound between Days 25 and 35 following insemination. viability decreased (P cO.05) from 59% at Day 7 to 27% at Day 14 in the hot season, but was not decreased during the cool season (52 vs. 60%). Pregnancy rate at Days 25 to 35 was 2 1% in the hot season, which was less (P < 0.05) than the 36% in the cool season. The percentage of unfertilized ova collected in both the hot and cool seasons suggests that fertilization failure was not affected by season of breeding. In summary, embryonic loss after Day 7 of pregnancy appears to be a problem in this hot, dry climate. Key words:

cattle, season, fertilization,

embryo mortality

Acknowledgments This manuscript was approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 91- 1I-5335. This research was supported by Masstock Research & Development International, Box 8524, Riyadh 11492, Saudi Arabia. “Current address: TEAGASA, Agriculture & Food Development Authority, Department of Dairy Husbandry, Moorepark Research & Development Division, Fermoy Co, Cork, Ireland. bReprint requests.

Copyright 0 1993 Butterworth-Heinemann

Theriogenology

720 INTRODUCTION

The dairy industry in Saudi Arabia is faced with problems associated with impaired reproductive performance under high environmental temperatures during the summer season (April through September) and a seasonal demand for milk and milk products, which peaks during the hot months of the year. To satisfy this market, the dairy industry aims for a peak calving period between January and April. To maintain the concentrated seasonal calving pattern, pregnancy rates to artificial insemination (AI) must be maximized during the hot summer season. It is important, therefore, to identify to what extent embryo mortality contributes to a recurring reduction in fertility for lactating dairy cattle in this adverse environment. Reproductive performance has been reported to be impaired in cattle under heat stress conditions (1,Z). Attempts to alleviate the associated problems have included environmentzd control (3,4), embryo transfer (5,6), and more recently, heat shock treatment of embryos (7). Using embryos harvested from superovulated cattle, Monty and Racowsky (8) and Putney et al. (9) have reported that most early embryo mortality in dairy cattle in a heat-stress environment takes place prior to Day 7 of gestation. Previously, Dutt (10) had reported that embryonic mortality is increased by heat stress both prior to and after mating in the ewe. Cattle exhibit a normal estrous cycle of 18 to 24 days if an embryo fails to inhibit natural luteolysis (11). How the bovine embryo prevents luteolysis is not completely understood. The viable bovine embryo has recently been shown to produce prostaglandin % between Days 6 and 12 (12) as well as bovine trophoblast protein-l @V-l) from Day 15 of gestation (13). More recently, Imakawa et al. (14) have identified bTP-1 as a member of the alpha-interferon family. Furthermore, the interestrous interval has been increased by infusing a recombinant alpha-interferon into nonpregnant cows (15). Failure of the embryo to produce sufficient bTP-1 to prevent luteolysis may contribute to early embryo mortality in gestating cattle. It has been proposed that the ability of the embryo to produce bTP-1 is dependent on embryo size (16). However, only bovine embryos greater than 15 mm in length between 15 and 17 days of age have been shown to produce bTP- 1. Biggers et al. (17) exposed beef cattle to either a thermoneutral environment (22°C) or to a heat-stress environment (33 to 37°C) between Days 8 and 16 of gestation. In this study, both luteal tissue and conceptus wet weights were reduced in the cows exposed to heat stress. A loss in the ability of these embryos to produce bTP-1 or other cellular products may help to explain the components of early embryo mortality that are associated with conditions of heat stress. The objectives of this experiment were 1) to identify the level and stage of embryonic mortality that occur in lactating dairy cattle in a hot, dry climate (Saudi Arabia) and 2) to compare the post-insemination embryo mortality rate in dairy cattle in a hot, dry climate with that in the same herds during the cooler, winter months.

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MATERIALS Experimental

AND METHODS

Animals and Environment

The experiment was replicated in both the cool and hot seasons of the year. The hot season component was conducted on three large dairy farm units (with a range of 1,200 to 1,600 lactating females per unit) during the period extending from May 28 to August 11, 1988. The cool season component of the study was conducted from December 15, 1988 to January 19, 1989. The dairy units were located between 130 and 240 km from Riyadh, Saudi Arabia, within the region of the Tropic of Cancer. During the warm season of this study, daily maximum temperatures ranged between 44 and 53”C, while daily minimum temperatures ranged between 24.5 and 35°C with low daily humidity. In the cool season, daily maximum temperatures ranged between 15 and 2O”C, while daily minimum temperatures ranged between -1 to 8°C with low to moderate daily humidity. During the study, the environmental conditions in this part of Saudi Arabia were consistent on a day-to-day basis, with little fluctuation in either the temperature or the humidity between artificial insemination and embryo collection. For the cows in this study, the previous breeding season had begun at the end of March and the peak calving period occurred between January and April. The cows were housed under either a spray and fan cooling system or a Korral Kool evaporative cooling system, as previously described by Ryan (18). The cows were fed a total mixed ration of corn silage and concentrate along with fresh alfalfa for an average daily feed intake of 21 kg of dry matter per day. All cows were milked three times daily in herringbone or rotary parlors. Estrus detection was conducted by experienced technicians three times or more during each 24 hours on all cows in the milking herds. Each female was artificially inseminated only once during the study with frozen-thawed semen from one of a battery of proven Holstein sires (n=22), using the standard a.m.-p.m. rule for insemination and the same cadre of inseminators. Experimental

Design

In this study, Holstein cows were partitioned into paired groups within farm and day of AI by post partum interval, current milk production and parity. Primiparous and multiparous females were each allocated separately within these parity groups. The females were then placed into paired subgroups of three, based on similar post partum intervals and current milk production recorded within 14 days of AI. The experimental animals were then allocated at random to three treatments within subgroups, which were partitioned within each season as shown in Table 1. The treatments consisted of Group I, nonsurgical embryo collection on Days 6 or 7 post insemination; Group II, nonsurgical embryo collection on Days 13 or 14 post insemination; and Group III, a control group in which embryos were not collected. The cool season was restricted to 3 weeks for embryo collection. To maximize the number of females for comparison, the animals were initially grouped on the day of AI as described above. However, during the second week prior to embryo collection, the females were allocated randomly to either Group II or Group III. In the first week

Theriogenology

722

immediately prior to and after the start of embryo collection, the females were allocated randomly to one of the three treatment groups on the day of AI. During the second week of embryo collection, the females were allocated randomly to either Group I or Group III on the day of AI. The result of this allocation accounts for the disparity in the number of females allocated to treatment groups in the cool season (Group I = 78, Group II = 75 and Group III = 106). Table 1. Experimental

design for embryo collection during the warm and cool seasons Day of embryo collection after mating Days 6 to 7 post AI

Season of the Year Hot summer months Group I Group II Group III Cool winter months Group I Group II Group III

Days 13 to 14 post AI

Embryos not collected AI Control” group

+ + +

+ + +

“Pregnancy was determined between 25 and 35 days post AI by ultrasonography. During the hot season, 107 females were allocated for nonsurgical collection in Group I and in Group II. Embryo collection was attempted from 93 females on Days 6 and 7 and from 97 females on Days 13 and 14. During the cool season, 78 and 75 females were allocated for nonsurgical embryo collection in Group I and Group II, respectively. Nonsurgical embryo collection was subsequently performed on 64 and 63 females in Group I and in Group II, respectively (Table 2). The disparity between the number of females that were allocated to embryo collection and the number actually performed was accounted for by females that were diagnosed as having cystic follicles or not having a detectable corpus luteum (CL) at the time of embryo collection. In both the hot and cool seasons, 106 females were allocated to Group III. All embryo collections were performed at the three dairy units by the same technician. During the hot season, 2 days per week were allotted to each dairy farm unit (five different weeks) to allocate females to control and embryo collection groups. During the cool season, allocation of females to treatment groups and subsequent embryo collections were restricted to a 21-day period for management reasons. Experimental

Procedure

All experimental females were artificially inseminated with frozen-thawed semen from fertile Holstein bulls. Any female diagnosed with the aid of ultrasonography as

Theriogenology

723

having cystic follicles on the day of AI was not allocated to treatment groups. At the time of AI, the diameter of follicles 2 10 mm and the total number of follicles per animal were measured and recorded by ultrasonography with a 5 Mhz probe (I_.S-lOO(ra; TokyoKeiki, Japan). In addition, labia pigmentation scores, rectal temperatures, respiration rates, body weights, body condition scores, uterine size scores, uterine tone scores and backfat measurements were recorded from a random sample of treatment females at the time of insemination. The labia pigmentation score consisted of a subjective value ranging from 1 (labia with white pigmentation) to 5 (labia with dark red appearance). The rectal temperature was based on a 2 to 4 minute reading. The respiration rate was recorded as the number of respirations per minute. The body weight of each female was calculated using a weighband (Dalton’s’, Wexford, Ireland). This was strapped tightly around the girth of the animal, and a reading was taken using the calibrated markers on the tape. Each female was given a body condition score using a procedure described by the British Ministry of Agriculture and Fisheries (19). Body condition scores ranged from 0 (severely emaciated) to 5 (excessive fat cover). The uterine size score assigned ranged from 0 to 5, in which 0 represented a small pelvic uterus and 5 represented a large uterus that extended beyond the pelvic rim. Uterine tone scores ranged from 1 to 5, with the largest number representing the greatest uterine tone. Backfat was measured using a standard ultrasonography procedure. The probe was placed between the hook and pins lateral to the tailhead, and the measurements were taken from the ultrasound video screen using calipers. Using the lifetime production records of the cows the following data were obtained: cow age, parity, number of services, average number of services, post partum interval, calving date, AI date, estrus to AI interval, projected 305-day milk yield and current milk yield. The post partum interval was calculated for each female as the number of days between the calving date and the date of AI. The average number of services was calculated using the post partum interval divided by the number of post partum services. The estrus to AI interval was calculated by subtracting the previous date of standing estrus from the treatment AI date. The projected 305-day milk yield and the current milk yield of the females were both recorded within 14 days following insemination. Embryo Collection If an animal was allocated for nonsurgical collection group and the cervix could not be dilated to pass the uterine catheter, this female was exchanged for a comparable animal in Group III. This exchange occurred on four occasions during this study. At the time of embryo collection, ovarian structures were evaluated by ultrasonography. The number of follicles and the number of corpora lutea were recorded. Nonsurgical embryo collection was performed from the uterine horn ipsilateral to the CL, using 240 to 300 ml of Dulbecco’s phosphate-buffered saline (Gibco, Grand Island, NY) supplemented with 0.2% bovine serum albumin (Gibco, Grand Island, NY) as the flushing medium.

Theriogenology

724

An l&gauge Foley catheter with a 30-ml ribbed balloon (Bardexo; C.R. Bard, Inc., Murray Hill, NJ) was used for nonsurgical embryo collection of the experimental animals. The catheter was inserted into the uterine horn with the aid of a stilette, and the balloon cuff was inflated with 5 to 10 ml of sterile water. The flushing medium (38°C) was introduced to the horn in aliquots of 30 to 60 ml. The recovered flushing medium was then passed through a 70-pm screen filter (EmCone, Veterinary Concepts, Inc., Spring Valley, WI) to harvest the embryos. The flushing medium retained in the filter after collection was examined for embryos using a Nikon inverted microscope (10 to 40X). Embryo Evaluation The Day-6 and Day-7 embryos were morphologically evaluated and assigned an embryo quality grade based on a scale from 1 to 4, using criteria similar to that previously described by Lindner and Wright (20). The embryos were classified as follows: Grade 1 = excellent, Grade 2 = good, Grade 3 = poor and Grade 4 = degenerating. The Day-13 and Day-14 embryos were also assigned embryo viability scores of 1 through 5. Embryos were classified as potentially viable at this stage if they had hatched from the zona pellucida and begun to elongate. Conceptus size (diameter or length) and cell morphology were also evaluated. Viability scores from 1 through 3 classified embryos as potentially viable, while those with a score of 4 were classified as nonviable. Embryos with a viability score of 4 were allocated to the embryonic loss category in this study. Statistical Analysis Chi-square analyses (21) were used to detect differen‘ces in embryo viability between the early embryo stages and the differences in pregnancy rates between the three stages of embryo development. Females diagnosed as having either cystic follicles or no functional luteal tissue at the time of collection were classified as nonpregnant, but were included in the analysis for comparison of pregnancy rates at different stages of gestation. Multiple regression and correlation analyses (22) were used to evaluate the effects of different variables monitored on the probability of a viable embryo being present at the time of collection or a conceptus at pregnancy diagnosis. For this type of analysis, one or more viable embryos at any stage of gestation were designated as a pregnancy. RESULTS During the hot and cool seasons, between 9 and 18% of the females allocated for embryo collection at either Days 6 to 7 or Days 13 to 14 after AI did not undergo the procedure (Table 2). At the time of the scheduled embryo collection these females were either diagnosed as having cystic follicles or did not have luteal tissue identified by ultrasonography. The percentage of females with one or more embryos recovered per collection was similar at both Days 6 to 7 (51.6%) and Days 13 to 14 (51.5%) in the hot summer months. However, the embryo recovery rate during the cool winter months on Days 13

Theriogenoiogy

725

to 14 (66.6%) tended to be higher than on Days 6 to 7 (53.1%), but these recovery rates were not significantly different (Table 2). In the hot season, the recovery rate was 5 1.5 % (981190) and was not different (P>O.lO) from the 59.8% (76/127) for females undergoing embryo collections in the cool season. Table 2. Summary of embryo collection data from dairy cows during the hot and cool seasons of the year Embrvo collection Season of the Year Hot summer months No. of animals artificially inseminated No. of animals not collected” % No. of animals collected % No. of animals with no ova/no. of embryos recovered % No. of animals with ova/no. of embryos recoveredb % Cool winter months No. of animals artificially inseminated No. of animals not collected” % No. of animals collected % No. of animals with no ova/no. of embryos recovered % No. of animals with ova/no. of embryos recoveredb %

Group I Days 6 to 7

Group II Days 13 to 14

107 14 13.1% 93 86.9%

107 10 9.3% 97 90.6%

45 48.4%

47 48.5%

48 51.6%

50 51.5%

78 14 17.9% 64 82.1%

75 12 16.0% 63 84.0%

30 46.9%

21 33.3%

34 53.1%

42 66.6%

“At the time of embryo collection, these females were either diagnosed as having cystic follicles or did not have a CL. bIncludes females with more than one ovum or embryo recovered during this season. In the hot season, 45.8% of the females from which embryos were collected on Days 6 to 7 had at least one viable embryo, and this was higher (P <0.05) than the 24% for females undergoing collection on Days 13 to 14 of gestation (Table 3). Furthermore, of the females with a single ovulation (at the time of collection) in the hot summer months, 44% (20/45) had a viable embryo on Days 6 to 7, and this was higher (I’ < 0.05)

726

Theriogenology

Table 3. Summary of embryo parameters during the hot and cool seasons of the year

Item

Days =

Females with ova/ embryos recovered Females with at least one viable embryo % Females with no viable embryos %

Season of the year Cool season Hot season Group I Grout II Group I ?E? 13-14 6-7 6-7

48

50

34

42

22 45.8%”

E.O%b

14 41.2%”

22 52.3%’

26 44.2%”

38 76.0Ab

20 58.8%”

20 47.6%’

Females with one oval embryo recovered 29 % ZO% 85.3%” E.746” Females with one viable embryo 11 20 % 44.4%” 24.4%b EO%’ 3?.0%” Females with no ovum or viable embryo 34 20 % 69.0%” :;.096a f: 5%” 75.5%b 4 Females with one of two UFO 7 9’ 8 % 27.6%” 10.5%a 20.0%’ 15.5%’ Females with one of two degenerating embryo 27 12 % :;.swa E.5%” 60.0%b 41.4%” ___________________________________-_________________________________________~~-~~~~~~~~~----Females with two ova/ embryos recovered 5 5 % 6!2%n 14.7%” i.5%’ 10.0%” Females with two viable embryos 1 3 2 % 75.0%” 6Z.6%” 20.0%’ 40.0%” Females with no viable embryos 2 % 2kO%’ 3:.3na s:.o%a 40.0%” Females with one of two viable embryos 0 1 % iO%” 0.0%’ 20.0%” ooo%a Females with one of two UFO 0’ 0’ % 0.0%” :.o%a :.o%’ o.o%a Females with one of two degenerating embryos % :.O%a i.O%l ELO%” 2ko%a ‘sbMeans within the same row and the same season with different significantly different (p
superscripts

are

Theriogenology than the 24% (1 l/45) for females from which a viable embryo was collected on Days 13 to 14. When the embryos from both single and multiple embryo recoveries were combined, the embryo viability scores decreased (P
Pregnancy rates for lactating females at different stages of gestation in the hot and cool seasons

Season of the Year Hot summer months No. of females/group No. pregnant % Cool winter months No. of females/group No. pregnant %

Days 6 to 7

Stage of gestation Days 13 to 14

Days 25 to 35

62’ 22 35.5”

59’ 12 20.3”

106 22 20.7”

48 14 29.2”

54’ 20 37.0b

106 38 35.8b

“*bMeans in columns with different superscripts are significantly different (P < 0.05). “Includes females that were either diagnosed as having cystic follicles or did not have detectable luteal tissue.

728

Theriogenology

Theriogenology The stage of embryo development was the principal factor affecting embryo viability in the multiple regression analysis for the hot season (Table 5). The probability of a viable embryo was greater (P < 0.05) for females in Group I when compared with subsequent periods of gestation for similar females. The parameter estimate for this variable was 0.28, indicating that 28% more females were predicted to have a viable embryo at either Day 6 or 7 when compared with the subsequent stages of embryo development. The number of CL also tended to have a positive effect on embryo viability (P < 0.08). The parameter estimate for this variable was 0.11. However, there was no evidence from either Chi-square analysis or Pearson correlation coefficients of an effect of the number of CL present on pregnancy rate, or that there was a relationship between the number of CL and pregnancy status during the hot season. Multiple regression analysis revealed (Table 6) that there was no evidence of an effect of the stage of gestation on the probability of a female having a viable embryo during the cool season. The number of CL had an effect on the probability of a viable embryo. The parameter estimate for this variable was 0.25, indicating that the estimated increase in viability was 25% for a unit increase in number of CL per female. The mean (fSEM) post partum interval and number of services for females artificially inseminated during the hot season were llOf4.17 days and 2.27fO.18 services, respectively. These means were less (P < 0.05) than the corresponding values of 178.7f6.0 days and 3.74f0.13 services for females inseminated during the cool season. However, there was no evidence of a correlation (P > 0.10) for embryo viability with either the number of services or post partum interval in both the hot and in cool seasons. Also, neither the number of services nor post partum interval was significant (P>O. lo), using a regression analysis for embryo viability in the hot and cool seasons (Tables 5 and 6). The only animal parameters evaluated that correlated with embryo viability score were body weight, body condition scores and respiration rates in the hot Season and the number of CL in the cool season. Both an increase in body weight (P=O.O7) and body condition scores (P=O.O8) of the females were positively correlated with embryo viability in the hot season. An increase in respiration rate during the hot summer season was negatively correlated with embryo viability in this study. As the number of CL per female increased in the cool season, there was a correlated increase in the probability (PlOmm) in the hot season, and this was less (P
Theriogenology

730

Table 5.

Multiple regression analysis of factors affecting embryo viability during the hot summer season

Source”

df

Group I Group II PPI NS ANS AIE BW NCL CMY NFOLAI

1 1 1 1 1 1 1 : 8;

Mean square error 1.06 0.12 0.03 0.03 0.09 0.10 0.06 0.60 0.16 0.22 0.20

F value 5.36 0.63 0.17 0.18 0.49 0.52 0.32 3.06 0.84 1.14

PLF 0.02 0.42 0.68 0.66 0.48 0.47 0.57 0.08 0.36 0.28

%roup I = embryo collection at Days 6-7, Group II = embryo collection at Days 13-14, PPI = postpartum interval, NS = number of services, ANS = average number of services, AIE = estrus interval prior to onset of treatment, BW = body weight, NCL = number of corpora lutes present at the time of embryo collection, CMY = current milk yield, NFOLAI = number of follicles present at the time of AI.

Table 6.

Multiple regression analysis of factors affecting embryo viability during the cool winter season

Source”

df

Group I Group II PPI NS ANS AIE BW NCL CMY NFOLAI AGE PYI PY2 PMY Error

1 1 1 : 1 1 1 1 1 1 1 1 12:

Mean square error 0.11 0.20 0.10 0.28 0.23 0.01 0.13 1.33 0.01 0.04 0.10 0.09 0.12 0.22 0.25

F value 0.45 0.79 0.41 1.13 0.91 0.02 0.05 5.25 0.02 0.14 0.40 0.39 0.49 0.86

PzF 0.50 0.37 0.52 0.29 0.34 0.90 0.88 0.02 .0.89 0.70 0.52 0.53 0.48 0.35

‘Group I=embryo collection at Days 6-7, Group II=embryo collection Days 13-14, PPI=postpartum interval, NS=number of services, ANS=average number of services, AIE=estrus interval prior to onset of treatment, BW=body weight, NCL=number of corpora lutea present at the time of embryo collection, CMY =current milk yield, NFOLAI=number of follicles present at the time of AI, AGE=age of thedonor female, PYl =tirst parity females, PY2=sewnd parity females, PMY =projected 305day milk yield.

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Ttieriogenology

the hot season, and this was not different from the 23.4% (60/256) of females examined during the cool season (Table 7). The recovery rate per CL was lower (P < 0.05) in the hot season (44.7%; 106/237) than in the cool season (56.3%; 85051). The recovery rate for females (n=248) with one CL (54.0%; 134/248) was higher (PcO.05) than for females(n=69)withmorethenoneCL(41.3%; 571138). Duringthehotseason, 55.5% Table 7.

Tbe frequency of ova/embryos numbers of CL in females

Season of the Year Hot season Group” I

CL/ donori’

No. of females( %)

and pregnancy

UFO’

associated

with different

Collection Total Viable embryos embryos( %)

PRd

1 2 3

67(72.0%) 24(25.8%) 2 (2.2%)

7 2 1

27 13 1

15(55.5%) 8(61.0%) l(lOO.O%)

-

II

1 2 3

78(80.4%) 19(19.6%) 0 (0.0%)

6 1 0

35 13 0

9(25.7%) 4(30.8%) 0 (0.0%)

-

III

1 2 3

51(77.3%) 14(21.2%) 1 (1.5%)

1 2 3

50(78.1%) 14(21.9%) 0 (0.0%)

8 0

19 12

9(47.4%) 7(58.3%)

-

II

1 2 3

53(84.1%) 10(15.9%) 0 (0.0%)

4 0

28 14

14(50.0%) 11(78.6%)

-

III

1 2 3

85(80.2%) 20(18.9%) 1 (0.9%)

Cool season Group I

12 5 0

30 8 0

“Group I=embryo collection at Days 6 or 7, Group II=embryo collection at Days 13 or 14, Group III= no embryo collection and pregnancy was diagnosed by ultrasound between Days 25 and 35 of gestation. bNumber of corpora lutea per female diagnosed by ultrasonography. ‘UFO=unfertilized ova. dPR=control females diagnosed pregnant in Group III.

732

Theriogenofogy

(1927) of the embryos recovered from females with one CL in Group I were viable compared with 64.3% (9/14) in females with multiple CL. In corresponding females in Group II, 25.7 (g/35) and 30.8% (4/13) of the embryos recovered from females with one and two CL, respectively, were considered viable. In the cool season, 47.4 (g/19) and 58.3% (7/12) of the embryos recovered from females in Group I with one and two CL, Corresponding females in Group II had 50.0 respectively, were considered viable. (14/28) and 78.6% (1 l/14) ) viable embryos harvested from females with one and two CL, respectively (Table 7). Overall, the viability of embryos recovered from females with one CL was 43.1% (47/109) and was less (PcO.10) than for females with more than one CL at (58.5%; 31153).

DISCUSSION In the current study, the embryo mortality rate increased between Days 6 and 14 of gestation during the hot season. These findings are in contrast to those of Monty and Racowsky (8) and Putney et al. (9), who reported that most embryo mortality occurred prior to Day 7 of gestation in dairy cattle managed in a hot climate. It should be noted that the results in both of the latter studies were based on embryos recovered from superovulated cattle 7 days following mating. The superovulation procedures may have imposed an additional stress on the animals, thus influencing the stage at which embryo mortality occurred. Superovulation treatments often alter the normal endocrine profile, which may cause premature ovulations (23,24), abnormal follicular steroidogenesis (25) and an early resumption of meiosis of oocytes (26). In addition, superovulation treatments have been associated with a high level of cytogenetically-detectable abnormalities in the embryos (27,28). The stages at which embryo mortality occurred under the hot climatic conditions in our study, may not be primarily attributable to heat stress, but to the overall discomfort of the animals. Although the daily maximum ambient temperatures ranged between 44 and 53°C in the hot season compared with 15 to 20°C in the cool season, the cows were not directly exposed to the elevated temperatures in the hot season and were housed in either Korral Kool or spray and fan evaporative cooling systems to maintain milk production. Under the Korral Kool system (4), the pregnancy rates of cows after AI at 40 to 60 days post partum were higher than those of cows maintained under the spray and fan system. We attributed part of this difference to the improved resting conditions that resulted under the Korral Kool system. In the early post partum period, the females need to adjust to the added heat load of lactation and the metabolic problems associated with energy intake needed to meet the demands of body maintenance and lactation (29). Ryan (unpublished data) noted that the pregnancy rates of cows in a hot climate inseminated between 42 and 90 days post partum were lower, if either a physiological problem was diagnosed or functional luteal tissue failed to form prior to 40 days post partum. Finally, it cannot be overlooked that the management systems employed in this hot climate may have influenced at which stage of development that embryonic mortality occurred. In the cool season of our study, most of the early embryo deaths occurred prior to Day 7 of gestation. In contrast, Diskin and Sreenan (30) and Roche et al. (31) have indicated that most embryo deaths in beef cattle occur between Days 8 and 18 of gestation in temperate climates.

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The fertilization failure rates reported in the present study were similar to those reported by Putney et al. (9) for dairy cattle under heat stress conditions. In the latter report, fertilization rates did not fall below 80% under conditions of elevated temperature. The lower incidence of unfertilized ova among collections at Days 13 to 14 in our present study may in part be explained by a breakdown of the unfertilized ova prior to their recovery at Days 13 to 14. The significant increase in embryo mortality between Days 6 and 14 of gestation in the hot season is similar to the finding reported by Diskin and Sreenan (30) and Roche et al. (31) for cattle in a temperate climate. In an earlier study conducted by this laboratory (7), Day-6 bovine embryos were cultured on oviduct cells at 40°C to mimic the chronic stress of an in vivo environment, and the embryos hatched earlier than the controls cultured at 38.6”C. However, significantly fewer embryos cultured at 40°C were viable at 60 hours after the onset of culture as a result of embryonic death after hatching (7). The increase in embryo mortality between Days 6 and 14 during the summer months in the present study, may be explained by increased metabolism of the embryo at an elevated uterine temperature prior to hatching. These findings differ from those of Dutt (lo), who reported that embryonic mortality increased markedly during the first week of gestation in ewes that had been heat-stressed shortly before and after mating. The pregnancy rates at Days 13 and 14 were similar to those between Days 25 and 35 in both the hot and cool seasons. Boyd et al. (32) also reported that embryo survival did not differ for dairy cows slaughtered at 12 to 16 days after mating compared with that of cows slaughtered at 25 to 26 days after mating. The bovine embryo can produce bTP1 starting at approximately 15 days of pregnancy, and this may prevent luteolysis thus resulting in extended luteal function (16). These findings and those of the present study imply that failure to prevent luteolysis, by embryonic signaling, may not be a contributory factor to embryonic mortality. The incidence of two or more ovulations was higher than expected for Holstein cows, but was in agreement with the high incidence of twin ovulations previously reported for Holstein cows in Saudi Arabia (33). Scanlon et al. (34) have reported that the incidence of twin ovulations was 2.5% for beef heifers and 3.3% for dairy cows in a temperate climate. The frequency of multiple ovulations was similar in both the hot and cool seasons in the present study. Under chronic stress conditions, one would expect the cycling female to exhibit erratic estrual behavior patterns until a more satisfactory environment arose. The kangaroo, for example, maintains its embryo in a state of diapause under the conditions of lactational stress that are associated with a young kangaroo in the pouch (35). The increase in multiple ovulations may result from a lack of dominance by a single follicle in the preovulatory follicular wave. In the present study, the embryo recovery rate per CL was reduced by an increase in the number of CL. When ewes were immunized against androstenedione, it was reported that the ovulation rate increased but the embryo recovery rate decreased 2 days after mating (36). The reduced recovery rate by an increased number of CL may be related to the failure of the fimbriae to collect the ova at the time of ovulation. Looney

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(personal communication, 1988) administered gonadotropins to 147 beef donor cattle, and on Day 2 after mating the ova were surgically recovered from the oviducts. The mean number of CL and number of ova per donor was 20.6 and 13.6, respectively, resulting in a 66% recovery rate of potential ova. The recovery rate per CL in our study was lower than expected, but was similar to the 44% recovery rate reported by Gordon et al. Ayalon (37) for similar type dairy cattle treated with gonadotropins for superovulation. et al. (38) reported that ova/embryo recovery rates for dairy cattle were 58% during cool seasonal periods and 38% during hot weather periods. These differences were attributed to failure of the fimbriae to pick up the ova, to degeneration of the ova in the oviduct, or to a possible disturbance in oviductal transport. Gordon et al. (39) have reported higher pregnancy rates in cattle than those achieved in the present study to a single mating after administering low dose levels of gonadotropins to increase the number of multiple ovulations. In contrast, Kidder et al. (40) have noted a detrimental effect of natural naturally occurring multiple ovulations in Holstein-Friesian cattle. The pregnancy rate was 28.6% for females with multiple ovulations compared with 57.5% for females with single ovulations. Also, Reynolds et al. (41) reported that all cows with more than six ovulations had degenerating embryos present at ~50 days of gestation. In the present experiment, the probability of a viable embryo being present was increased by the presence of more than one CL during the cool season; however, this was not evident in the hot season. In conclusion, embryonic death prior to Day 7 was similar in both the hot and cool seasons. During the hot season, embryonic death increased markedly between Day 7 and Day 14 of pregnancy. There may be the potential for reducing embryonic death at this stage of development by heat shock treatment of bovine embryos in vitro (7), prior to transfer to recipient females. Such studies are presently underway at this station. Alternatively, it may be possible to avoid embryonic death at this stage of development by transferring of Day-10 to Day-14 embryos to recipient animals. Retteridge et al. (42) reported that acceptable pregnancy rates in cattle could be achieved following the transfer of embryos up to Day 16 post mating. Recent developments in bovine in vitro fertilization (43) and co-culture procedures (44,45) may enable embryos derived from in vitro fertilization to develop in vitro to the 1Cday stage of development for subsequent transfer to recipients. The frequency of natural multiple ovulations for the dairy cattle in the present study was higher than what would be normally expected. However, we found no evidence that the increase in ovulation rate would increase the probability of a viable embryo during the hot summer months, when pregnancy rates from AI are lower than during the cool months. REFERENCES 1. Stott, G.H. Female and breed associated with seasonal fertility variations in dairy cattle. J. Dairy Sci. &1698-1704 (1961). 2. Ingraham, R.H., Gillette, D.D. and Wagner, W.D. Relationship of temperature and humidity to conception rate of Holstein cows in subtropical climate. J. Dairy Sci. 521476-481 (1974).

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