A. K. McNeel

TRIENNIAL REPRODUCTION SYMPOSIUM: Limitations in uterine and conceptus physiology that lead to fetal losses12

Conceptus losses in livestock occur throughout gestation. The uterus and the embryo-placenta-fetus play interconnected roles in these losses, the details of which depend on the period of gestation and the species. Studies in sheep and pigs have indicated that the uterine glands are essential for full fertility, based on experiments where gland development was reduced through the use of exogenous hormones. In sheep and cattle, normally the uterus is well able to support more than a single fetus although these species differ in the consequences of multiple births. When 2 conceptuses are present, the placentas of cattle often anastomose, putting 1 fetus at risk if the other is lost. One likely reason this does not occur in sheep is because sheep embryos undergo intrauterine migration, similar to pigs. In pigs, the relatively equidistant separation of conceptuses is likely to be essential for optimizing conceptus survival as is the simultaneous and uniform elongation of blastocysts that occurs during the time of maternal recognition of pregnancy. Other studies in pigs have indicated that the size of the uterus influences litter size and therefore fetal losses. In response to crowded intrauterine conditions in the pig, increased conceptus losses begin to occur between d 30 and 40 of pregnancy, and further losses occur sporadically during later gestation. There is evidence that improved fetal erythropoiesis can reduce these losses. Other studies indicated that profound changes in placental development occurred under crowded intrauterine conditions that may contribute to losses during late gestation. Reductions in placental stroma formation may compromise the ability of the pig placenta to adapt to reduced uterine space. Consistent with this, both hyaluronan and hyaluronidase activity are decreased in the placentas of small compared with large fetuses. These results indicate that improvements in placental stroma formation could improve placental ability to compensate for reduced intrauterine space, resulting in increased placental function and reduced fetal losses during late gestation.

The consequence of level of nutrition on heifer ovarian and mammary development12

Replacing cows in the herd is second only to nutrition as the single greatest input cost in cow/calf beef production. The increased availability of cereal grains for feeding livestock has allowed replacement heifers to enter the production system at younger ages. Many heifer development programs feed to ensure heifers reach puberty before the time that they are mated to calve at 2 yr of age. Nutrition level during development has been associated with altered milk production and stayability. We hypothesized that heifers exposed to a lower nutrition level during the peripubertal period would have less methylation of the DNA in the mammary gland and ovarian cortex. We also hypothesized that the ovarian reserve would decrease in heifers fed for rapid growth compared to heifers fed for slow growth during puberty. At 257±1 d of age, heifers in the Stair-Step treatment (n=6) received 157 kcal ME/BW kg0.75 for 84 d and heifers in the Conventional treatment (n=6) were offered 228 kcal ME/BW kg0.75. At d 84, heifers were fed for an additional 83 d. Stair-Step heifers were offered 277 kcal ME/BW kg0.75, and heifers on the Conventional treatment received 228 kcal ME/BW kg0.75. Mammary weights (P=0.43), capillary area density (P=0.74), and capillary surface density (P=0.18) did not differ between treatments and neither did alveolar number (P=0.55) and alveolar density (P=0.49). Reproductive tract weights (P=0.69) and ovarian weight (P=0.68) and ovarian size (P>0.75) did not differ between treatments. In histological sections, Stair-Step heifers had more primordial follicles than Conventional heifers (P=0.02), but primary (P=0.59) and secondary (P=0.15) follicles did not differ. Global methylation of parenchymal tissue (P=0.82), mammary fat pad (P=0.45), and ovarian cortex (P=0.14) did not differ between treatments. Anterior pituitary weight did not differ between treatments (P=0.16). Our hypothesis that modifying peripubertal nutrition modifies global methylation of the mammary and ovary is not supported; however, our hypothesis that it modifies the ovarian reserve is supported.

Influence of puberty and antral follicle count on calving day in crossbred beef heifers

Lifetime productivity and longevity are greater in beef cows that give birth early in their first calving season. The ability of heifers to conceive early in the breeding season is traditionally thought to be a function of pubertal status; however, recent evidence suggests that antral follicle count is positively associated with calving day in pubertal beef heifers. Additionally, there is evidence to suggest that the total number of ovarian follicles may alter response to hormonal synchronization protocols. The objectives of this study were to confirm the beneficial influence of antral follicle count on calving day in beef heifers and to determine whether response to PGF2α is associated with differences in antral follicle counts. A 2 × 2 factorial experiment was designed to investigate the interaction between calving period (early vs. late) and PGF2α (control vs. PGF) on calving day and antral follicle count in yearling beef heifers (n = 95). As intended, calving day was less in the early calving period compared to the late calving period (P < 0.01). There were no differences in calving day in response to treatment with PGF2α (P > 0.05). There was a significant interaction between calving period and PGF2α on age at first calving (P < 0.01), such that heifers treated with PGF2α that gave birth early were younger than heifers treated with saline that gave birth early. Calf weaning weights were greater in the early calving group than in the late calving group (P < 0.01). Heifers that gave birth in the early calving group possessed more antral follicles at prebreeding ultrasonographic examination than heifers that gave birth in the late calving group (P = 0.05). These findings confirm that antral follicle counts are associated with calving day in pubertal beef heifers. The use of antral follicle counts as a prebreeding phenotype provides additional utility to reproductive tract scoring for commercial production because of its association with calving day. As a prebreeding ovarian phenotype, antral follicle counts may hold additional applicability for organic beef producers looking to reduce the length of their calving season without increasing the total number of replacement heifers retained.

A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight

The use of genetic markers to aid in selection decisions to improve carcass and growth characteristics is of great interest to the beef industry. However, it is important to examine potential antagonistic interactions with fertility in cows before widespread application of marker-assisted selection. The objective of the current experiment was to examine the influence of 2 commercially available markers currently in use for improving carcass traits, the myostatin (MSTN) F94L and μ-calpain (CAPN1) 316 and 4751 polymorphisms, on heifer development and reproductive performance. In Exp. 1, beef heifers (n = 146) were evaluated for growth and reproductive traits over a 3-yr period to determine if these polymorphisms influenced reproductive performance. In Exp. 2, heifers representing the 2 homozygous genotypes for the MSTN F94L polymorphism were slaughtered on d 4 of the estrous cycle and reproductive tracts were collected for morphological examination. In Exp. 1, there was a tendency (P = 0.06) for birth BW to be affected by MSTN with the Leu allele increasing birth BW in an additive fashion. Additionally, MSTN significantly affected the proportion of pubertal heifers by the start of the breeding season (P < 0.05) with the Leu allele additively decreasing the proportion pubertal; however, this did not result in a delay in conception or a decrease in pregnancy rates during the first breeding season (P > 0.15). The GT haplotype of CAPN1, which was previously associated with decreased meat tenderness, was associated with an additive decrease in birth BW of the first calf born to these heifers (P < 0.05). In Exp. 2, there were no differences between the MSTN genotypes for gross or histological morphology of the anterior pituitary, uterus, or ovaries (P > 0.05). From these results, we concluded that the MSTN F94L and CAPN1 polymorphisms can be used to improve carcass traits without compromising fertility in beef heifers. The influence of these markers on cow performance and herd life remains to be determined. While the delay in puberty associated with the MSTN F94L polymorphism did not negatively impact reproductive performance in heifers, caution should be used when combining this marker with other markers for growth or carcass traits until the potential interactions are more clearly understood.

Beef heifers with diminished numbers of antral follicles have decreased uterine protein concentrations

Previous research demonstrated a favorable relationship between the number of follicles detectable in the bovine ovary by ultrasonography and fertility, and bovine females with diminished numbers of antral follicles had smaller reproductive tracts. Therefore, we hypothesized that uterine function would be compromised in beef heifers with diminished numbers of antral follilcles. Angus heifers (n=480) were submitted for ultrasonographic evaluation of antral follicle number at 325 and 355d of age. After the second ultrasonographic examination, 40 pubertal heifers with the greatest average number of antral follicles (30.9±0.7) and 40 pubertal heifers with the lowest average number of antral follicles (14.2±0.7) were synchronized with two i.m. injections of prostaglandin F2α (25mg) administered 11d apart, and heifers were slaughtered on d6 (n=26 heifers/group) or d16 (n=14 heifers/group) of the resultant estrous cycle. The uterus was weighed, flushed for determination of protein content, and representative samples were fixed for determination of endometrial gland morphometry. Heifers in the Low group had fewer surface antral follicles and smaller reproductive tracts than heifers in the High group (P<0.01). Protein content of the uterine flushes was decreased in heifers in the Low group (P<0.01); however, there was no difference in the percent area of the endometrium occupied by endometrial glands. From these results, we conclude that the uterine environment of beef heifers with diminished numbers of antral follicles is less conducive to supporting early embryonic survival.

Estimates of epistatic and pleiotropic effects of casein alpha s1 (CSN1S1) and thyroglobulin (TG) genetic markers on beef heifer performance traits enhanced by selection1234

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC II) was subjected to marker assisted selection for 2 yr to equalize and genetic marker frequencies to evaluate the epistatic and pleiotropic effects of these markers on BW, reproduction, and first calf performance traits in replacement beef females ( = 171) managed under 2 postweaning development protocols. Traits evaluated on the heifers were birth BW, weaning BW, 11-mo BW, 12-mo BW, 13-mo BW, first breeding season pregnancy evaluation BW, first calving season BW, 11-mo puberty, 12-mo puberty, 13-mo puberty, first breeding season pregnancy, and first calf weaning rate. Additionally, heifers first calf performance traits of ordinal calving date, first calf birth BW, and first calf weaning BW (with and without age adjustment) were analyzed. Selection to increase minor allele frequencies and balanced sampling across genotype classes enhanced the ability to detect all genetic effects except dominance × dominance epistasis. The × genotype effect was significant ( < 0.05) for 11-mo BW and 12-mo BW and tended to be significant ( = 0.08) for 13-mo BW. Consistently, for all 3 traits, the most significant effect among epistatic × genotype effects was the additive effect, with the G allele decreasing BW. There were no associations between × genotype and fertility related traits ( ≥ 0.46) in this study. Additionally, there were no × genotype associations with first progeny performance traits ( ≥ 0.14). The large effect of the additive × additive interaction on first calf weaning BW was imprecisely estimated, which may warrant further investigation.

Postweaning nutritional programming of ovarian development in beef heifers

Peripubertal caloric restriction increases primordial follicle numbers at breeding, which may improve reproductive potential. Our hypothesis was that feed restriction was changing primordial follicle number through stimulation of follicle formation via leptin, roundabout axon guidance receptor, homolog 4 (), or or through inhibition of follicle activation via anti-Müllerian hormone (). Heifers ( = 30) were fed a ration consisting of 30% alfalfa hay, 69.8% corn silage, and 0.2% salt as DM. Heifers received the control diet for 42 d before an initial 6 heifers were ovariectomized at 8 mo of age. The remaining 24 heifers were divided into 2 treatment groups. Controls were offered 97.9 g DM/kg BW over the entire feeding period. Stair-step heifers received 67.4 g DM/kg BW for 84 d. Following the 84-d restriction, heifers were stepped up to receive 118.9 g DM/kg BW over a 15-d period and were held at this feeding level 68 d. At the end of the feed restriction (11 mo of age), ovaries were collected from 6 heifers per treatment, and at the end of the refeeding period (13 mo of age), ovaries were collected from 6 heifers per treatment. Plasma leptin concentrations were greater in control heifers than in stair-step heifers at 11 mo of age ( < 0.0001). In histological sections, stair-step heifers had more primordial follicles ( = 0.03) than control heifers at 13 mo of age. There was no difference in secondary or antral follicle numbers between dietary treatment groups or ages. Relative abundance of mRNA in ovarian cortex of control heifers was greater at 13 mo than at 11 mo or before feed restriction (8 mo; = 0.01). Relative abundance of mRNA in stair-step heifers at 13 mo was greater than before feed restriction ( = 0.02) and at 11 mo did not differ from 8 or 13 mo ( = 0.70). Relative abundance of mRNA in the ovarian cortex followed a similar pattern, being greater in stair-step heifers at 11 mo compared with control heifers ( = 0.001). At 13 mo, mRNA did not differ between treatments ( = 0.30). Abundance of mRNA in the ovarian cortex did not change due to dietary treatment or age ( > 0.10). In conclusion, developing heifers on a stair-step compensatory growth scheme resulted in larger ovarian reserve before the onset of breeding, which may have beneficial effects on increasing reproductive lifespan.

Technical Report: Serial collections of placentomes during parturition in cattle and subsequent reproductive performance1

Placental separation is a complex physiological event in reproductive physiology and the underlying molecular mechanisms remain unclear. When comparing different experiments the timing of tissue collections is a significant consideration due to the variability in time between fetal expulsion and expulsion of the placenta (30 min to >24 h). This makes comparison of tissues samples across animals difficult and supports the need for serial tissue collections within animal. Additionally, the instrument most commonly used, a modified Richter-Resinsinger effeminator, for placentome collections is difficult to obtain and there are no data in the literature record regarding subsequent reproductive performance of animals subjected placentome collections. To facilitate continued research into the physiology behind placental separation, we designed an instrument from readily available components and performed serial transvaginal placentome collections in cattle. Three placentomes at 2-h intervals were collected after expulsion of the calf in 18 multiparous cows. There was no incidence of mortality and all cows resumed estrous after the procedure. Neither time from placentome collection nor age had a significant effect on pregnancy status at diagnosis (P > 0.05). These results demonstrate the viability of and utility of this device for collecting multiple placentomes in cattle.

μ-Calpain (CAPN1), calpastatin (CAST), and growth hormone receptor (GHR) genetic effects on Angus beef heifer performance traits and reproduction

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.5-3.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1 wk difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection and breeders should not be concerned with how these markers affect reproduction and other heifer traits with the possible exception of CAPN1 effects on calving date.

144 EFFECT OF ANTRAL FOLLICLE COUNT IN BEEF HEIFERS ON IN VITRO FERTILIZATION/PRODUCTION

Our objective has been to compare the IVF and in vitro production (IVP) of embryos from low and high antral follicle count (AFC) heifers. This is the fourth year of the study with years 1 to 3 reported individually. For this report, we add data for the fourth year and present a combined analysis (years 1 to 4) for the first time. Each year, AFC was determined on ~120 Angus heifers using transrectal ultrasonography. Ten heifers with the lowest AFC and 10 heifers with the highest AFC and all with evidence of oestrous cyclicity were synchronized with two 5-mL injections of PGF2α 11 days apart. Half were harvested on Day 5 to 6 and half on Day 15 to 16 of the oestrous cycle. The IVF procedure was slightly modified each year. For year 4, the IVF procedure included protocols for semi-defined media and was as described (IVP Protocol, P. J. Hansens Laboratory, University of Florida). Cumulus-oocyte complexes (COC) from follicles less than 8mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24h. Matured COC were fertilized using thawed frozen semen from a bull that was purified using isolate. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1×106 spermatozoa permL. About 24h later, presumptive zygotes were placed in micro drops of development medium under oil, and cultured (5% CO2; 5% O2; balance N2; 38.5°C). On Day 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of year (1 to 4), group (high or low AFC), and their interaction. The year×group interaction was not significant (P>0.10). Low AFC heifers, compared with high AFC heifers, had fewer numbers of COC (P<0.0001; 12.8±1.83v. 31.9±1.86), fewer numbers of COC that cleaved (P<0.0001; 8.0±1.38v. 21.6±1.40), and fewer numbers of COC that developed to the blastocyst stage (P<0.0001; 1.7±0.58v. 5.7±0.58). Year affected the numbers of COC that cleaved (P<0.003) and the numbers of COC that developed to the blastocyst stage (P<0.0001). Year also influenced the percentage of COC that cleaved (P<0.0002) and the percentage of COC that developed to blastocysts (P<0.0001). Group (AFC) did not influence (P>0.19) the percentage of COC that cleaved (61.2±2.83v. 66.4±2.83%, for low v. high AFC, respectively). Low AFC heifers had a lower (P<0.002) percentage of COC that developed to blastocysts (10.3±1.52%) than high AFC heifers (17.6±1.52%). These results indicate that high AFC heifers, compared to low AFC heifers, have more COC recovered, more COC cleaved, and more COC developed to the blastocyst stage. The percentage of COC cleaved did not differ between AFC groups; however, the percentage of COC that developed to the blastocyst stage was greater for high than low AFC heifers. This suggests a potential advantage in maternal to embryonic transition for high compared with low AFC heifers.