R.W. Rorie

Application of electronic estrus detection technologies to reproductive management of cattle.

Artificial insemination and embryo transfer programs are dependent on efficient and accurate detection of estrus. Visual observation is accurate at detecting animals in estrus, but efficiency ranges from approximately 50 to 70%. Electronic technologies have been developed in attempts to improve estrus detection efficiency. Commercially available electronic devices for estrus detection are based on changes in physical activity (pedometers), changes in electrical resistance of reproductive tract secretions (intravaginal resistance probes) or mounting activity (mount detectors). All of the commercially available electronic estrus detection devices can improve the efficiency of estrus detection in cattle. Pedometers are most applicable to lactating dairy cattle and have greater accuracy and efficiency when combined with visual observation. Intravaginal resistance measurement is perhaps the least practical method of estrus detection because of labor and animal handling requirements. Individual resistance measurement may have practical application for confirming other inconclusive signs of estrus. Mount monitors have the broadest application to beef and dairy cattle. HeatWatch, the only real-time radiotelometric system available, requires the least labor and animal handling and provides data on the time and duration of each mount. The less expensive stand-alone mount monitors also provide the necessary information for optimum timing of insemination and embryo transfer, but are more labor intensive.

EFFECT OF TIMING OF ARTIFICIAL INSEMINATION ON SEX RATIO

For a number of years, the time of insemination or mating during estrus has been believed to influence the sex ratio of offspring, with early insemination resulting in more females and late insemination, more males. Possible mechanisms of altering the sex ratio include facilitating or inhibiting the transport of either X- or Y-chromosome-bearing sperm through the reproductive tract, preferential selection of sperm at fertilization, or sex-specific death of embryos after fertilization. In livestock species, there is evidence for preferential selection of X- or Y-bearing sperm, based on the maturational state of the oocyte at fertilization. In deer and sheep, early and late insemination appears to skew the sex ratio toward females and males, respectively. In cattle, conflicting reports on the effect of time of insemination on sex ratio make the premise less clear. Many of the published studies lack adequate observations for definitive conclusions and/or are based on infrequent observations of estrus, making it difficult to assess the effect of time of insemination on sex ratio. It is likely that any effect of time of insemination on sex ratio in cattle is relatively small. Evidence is accumulating that treatments used for synchronization of estrus or ovulation in cattle may influence the sex ratio.

REPRODUCTIVE RESPONSES TO GRAZING ENDOPHYTE-INFECTED TALL FESCUE BY POSTPARTUM BEEF COWS

The objective was to determine pregnancy rate and stage of embryonic loss in response to grazing endophyte-free (E-; n = 20) or infected (E+; n = 30) tall fescue in postpartum beef cows with calves. Three weeks before estrus synchronization, cow-calf pairs were introduced to pastures (April 1999). Cows were synchronized and bred by AI after detected estrus for a period of 6 d and then by natural service for 62 d. Bulls were rotated weekly to minimize effects of fescue toxicosis on male fertility. Fetal development was monitored weekly between 30 and 60 d of pregnancy and at weaning using transrectal ultrasound. Respiration rate (52.0 +/- 1.4 vs 46.6 breaths/min; P < 0.02) and rectal temperature (39.6 +/- .09 vs 38.8 +/- .12 degrees C; P < 0.001) increased in E+ cows and serum concentrations of prolactin (7.2 vs 57.4 +/- 4.4 ng/mL; P < 0.001), total cholesterol (123.2 vs 149.6 +/- 3.6 mg/dL; P < 0.001), body condition (3.8 vs 5.2 +/- 0.15; P < 0.001; 1 = thin, 9 = fat) and adjusted weaning weight of calves (195.8 vs 210.8 +/- 4.5 kg; P < 0.02) were reduced compared to that of E- cows. Differences were not detected (E- vs E+) for estrus detection rate (84.9 +/- 10.6% vs 80.2 +/- 8.4%), pregnancy rate to synchronized estrus (41.7 +/- 11.8% vs 46.8 +/- 9.5%), overall pregnancy rate 30 d postbreeding (93.8 +/- 6.2% vs 93.5 +/- 5.1%), overall pregnancy rate at 60 d postbreeding (86.7 +/- 10.1% vs 81.2 +/- 8.3%), or serum concentrations of progesterone on day of PGF2alpha treatment (4.5 +/- 0.7 vs 4.5 +/- 0.8 ng/mL). Pregnancy losses that occurred between 30 and 60 d gestation were 6.0 (E-) vs 15.0 (E+) +/- 8.0% (P > 0.10) and occurred after environmental temperatures rose above 37.8 degrees C for three weeks. Total pregnancy losses that occurred by weaning (between 70 and 126 d of gestation) were 5.5 (E-) vs 17.6 (E+) +/- 8.0% (P > 0.10). Pregnancy rate and embryonic losses were not different between cows grazing E- and E+ tall fescue under these management conditions.

Effect of timing of artificial insemination on gender ratio in beef cattle.

It was recently reported that cows inseminated at approximately 10 or 20 h before an expected ovulation deliver predominately a bull or heifer calf, respectively. The objective of this study was to further investigate the effect of timing of insemination on the gender of offspring in cattle. Angus heifers (n = 41) and cows (n = 98) were used in the study. Heifers were synchronized with a 16-d treatment of melengestrol acetate followed 17 d later with an injection of PGF2alpha. Cows were synchronized with GnRH followed 7 d later with PGF2alpha. A HeatWatch electronic estrus detection system was used to determine the onset of estrus. Based on previous studies, it was assumed that ovulation occurs approximately 32 h after the onset of estrus. Therefore, animals were artificially inseminated at either 8 to 10 h (early; > or = 20 h before expected ovulation) or 20 to 25 h (late; < or = 10 h before expected ovulation) after the onset of estrus. Sixty to 80 d after insemination, ultrasonography was used to confirm pregnancy status and to determine the gender of fetuses. Gender of calves was subsequently confirmed at calving. Data were analyzed for effects of time of insemination and sire or semen batch on gender ratio, as well as any effect of length and/or intensity of estrus on conception rate and gender ratio. Twenty-nine of 41 heifers and 69 of 98 cows were detected in estrus after synchronization and were inseminated; 20 of 29 heifers and 48 of 69 cows were subsequently confirmed pregnant. Neither the length of estrus nor its intensity (number of mounts) had an effect on pregnancy rate or gender ratio (P > or = 0.418). Timing of insemination (early versus late) had no effect on gender ratio (P = 0.887). Semen from 13 sires representing 17 lots was used to inseminate the cows and heifers. No differences (P = 0.494) were detected in the gender ratios resulting from different sires or semen batches. In contrast to previous findings, our results indicate that inseminating beef cattle at approximately 20 or 10 h before an expected ovulation does not alter the gender ratio of the resultant calves.

Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP+BOEC; 3) in TALP+PHE; or 4) in TALP+BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.

Changes in ovarian function in mature beef cows grazing endophyte infected tall fescue

The objective was to examine follicular and luteal development and function in mature, lactating beef cows grazing endophyte free (E-) or endophyte infected (E+) tall fescue during the early postpartum period. Angus, Hereford, and Angus x Hereford cows were exposed to pasture for 37-39 days before synchronized estrus. Serum concentrations of prolactin were evaluated during the luteal phase before the synchronized estrus. Every Monday, Wednesday, and Friday for one estrous cycle ovaries were monitored by transrectal ultrasonography and blood was collected for determination of serum concentrations of progesterone and estradiol in cows that responded to synchronization. Signs of fescue toxicosis in E+ cows included decreased serum concentrations of prolactin (84.9+/-13.6 pg/ml versus 32.3+/-12.0 pg/ml; P < 0.009) measured during the luteal phase (day 37 of grazing) and decreased body condition of cows and weight of cows and calves (P < 0.001). Neither serum concentrations of progesterone or estradiol, nor diameter of the CL differed between treatments. Diameter of the largest follicle tended to be smaller for cows grazing E+ fescue, especially between days 8 and 12 of the estrous cycle (P < 0.08). Numbers of class 1 (3-5 mm) and class 3 (>10 mm) follicles were similar (P > 0.05) between treatments, but number of class 2 (6-9 mm) follicles was reduced in E+ cows for most of the cycle (days 10 through 20; P < 0.03). Length of synchronized estrous cycle, days open, calving interval, and pregnancy rate at 30, 45, 60, and 90 days post-breeding was similar (P > 0.05) among treatment groups. Even though follicular dynamics (diameter of the largest follicle and number of class 2 follicles) were altered in cows grazing E+ tall fescue, follicular function was apparently not affected by ergot alkaloids.

In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate

The effect of bovine serum albumin (BSA) lots on the development of in vitro-derived bovine embryos in synthetic oviductal fluid was investigated. Citrate concentration was determined for each lot of BSA, and then correlated with differences noted in the ability of BSA lots to support embryo development. Development of bovine embryos to the blastocyst stage was also compared after culture in chemically-defined medium with varying levels of citrate. There were distinct differences in the ability of the different BSA lots to support embryo development to the blastocyst stage (P<or=0.05). Citrate content (based on 3.2% BSA) of the medium varied from 320 to 1280 microM. Although there was an overall linear trend (P<0.001) for increased number of blastocysts with increasing concentrations of citrate in the medium, there were also substantial deviations (P=0.024) from this linear trend, suggesting that factors other than citrate could be responsible for stimulating blastocyst development. In the second experiment, the percentages of cleaved embryos that developed to the blastocyst stage in defined medium with 0, 100, 300 or 900 microM citrate ranged from 18.2 to 27.8% and were similar among treatments (P=0.441). The mean number of cells in the embryos developing to the blastocyst stage did not differ among treatments (P=0.545). Overall, these results indicate citrate has little affect on development of bovine embryos to the blastocyst stage in vitro.

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P=0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P<or=0.066). The number of blastocysts was increased by the use of serum instead of BSA in cumulus cell co-culture medium (P<0.001). Regardless of culture method, a similar percentage of embryos were excellent or good quality when BSA was used in medium (P>or=0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P=0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P<0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P=0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P>or=0.535).

Evaluation of permeating cryoprotectants for the cryopreservation of bovine trophoblastic vesicles

Abstract In the first experiment, bovine trophoblastic vesicles (bTV; 46/treatment) were exposed to 1.5 M glycerol, dimethyl sulfoxide, propylene glycol or ethylene glycol for 30 min at room temperature (27° C). Cryoprotectants were removed from bTV by sequential exposure to 0.75 M of the appropriate cryoprotectant plus 0.75 M sucrose for 10 min, followed by 0.75 M sucrose for 10 min. The bTV were then cultured in RPMI 1640 medium and evaluated daily (for 3 d) for morphological quality. Post-exposure morphological quality of bTV indicated ethylene glycol was the least toxic cryoprotectant to bTV, followed by glycerol, propylene glycol and dimethyl sulfoxide. In the second experiment, bTV (60 bTV/treatment) were exposed to the same cryoprotectants for 30 min, loaded into 0.25-ml plastic straws (3 bTV/straw) and frozen, using a bovine embryo protocol (1° C/min from 20° C to −6° C; seeded and held at −6° C for 10 min; 0.3° C/min to −28° C; 0.1° C/min to −35° C; plunge into liquid nitrogen). After a few days storage, the straws were allowed to thaw in air (27° C), the cryoprotectants were removed as described in Experiment 1 and the bTV were placed into culture. Daily evaluation for 3 d post-thawing indicated bTV cryopreserved in ethylene glycol were of higher morphological quality than those cryopreserved in either glycerol, propylene glycol or dimethyl sulfoxide. The post-thaw viability of bTV (20 bTV/treatment) cryopreserved in either 1.0 M, 1.5 M or 2.0 M glycerol or ethylene glycol was compared in the third experiment. The bTV were cryopreserved, thawed, cultured and evaluated as described for Experiment 2. Post-thaw morphological quality was highest for bTV cryopreserved in 1.5 M ethylene glycol, followed by 1.0 M ethylene glycol, 1.5 M glycerol, 2.0 M ethylene glycol or glycerol and 1.0 M glycerol, respectively. The results of this study indicate that ethylene glycol is less toxic to bTV than the other cryoprotectants evaluated. Based on post-thaw morphological quality, 1.5 M ethylene glycol is the cryoprotectant of choice for bTV.