Virgin Heifers Research Articles

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

Asociación entre características reproductivas y producción de leche, en novillas mestizas

This research was carried out in heifers from a crisscrossing program between theredHolstein and Brahman breeds, inVenezuela. (Co)variance components were estimated and the relationship between milk yield (P305), age (AGE1) and fertility at first service (FERT1) in virgin heifers was investigated. The analysis methodology was restricted maximum likelihood (REML),under a multivariate animal model. The mixed model included: the random effect of the animal; and the fixed effects of racial group, year-seasons of the event occurrence; for P305, the duration of lactation was also included as a covariate in quadratic form. Heritabilities were: 0.498 ± 0.022; 0.436 ± 0.020 and 0.024 ± 0.008, for P305, AGE1and FERT1 respectively. Genetic correlations between: P305-AGE1; P305-FERT1 and AGE1-FERT1, were: -0.281±0.040; 0.238 ± 0.117 and -0.076 ± 0.123, these results suggest a favorable genetic association between P305-AGE1, as well as,between P305-FERT1. Weighted regression analysis of the averages breeding valueson years, showed an improvement in P305 of 10.85 kg/year and a reduction of -0.1150 month/year in AGE1 representing 0.60 and 0.41% of the population means for those traits (p < 0.01);the annual genetic change for FERT1 was not significant (p > 0.05). The phenotypic changes in P305 and AGE1were not significant (p > 0.05). It is concluded that there is no genetic antagonism between P305 and FERT1 and that it is possible to reduce AGE1 for decreasing the costs for generating replacement heifers, improving the rate of genetic change in P305.

The Nasopharyngeal, Ruminal, and Vaginal Microbiota and the Core Taxa Shared across These Microbiomes in Virgin Yearling Heifers Exposed to Divergent In Utero Nutrition during Their First Trimester of Gestation and in Pregnant Beef Heifers in Response to Mineral Supplementation.

In the present study, we evaluated whether the nasopharyngeal, ruminal, and vaginal microbiota would diverge (1) in virgin yearling beef heifers (9 months old) due to the maternal restricted gain during the first trimester of gestation; and (2) in pregnant beef heifers in response to the vitamin and mineral (VTM) supplementation during the first 6 months of pregnancy. As a secondary objective, using the microbiota data obtained from these two cohorts of beef heifers managed at the same location and sampled at the same time, we performed a holistic assessment of the microbial ecology residing within the respiratory, gastrointestinal, and reproductive tract of cattle. Our 16S rRNA gene sequencing results revealed that both α and β-diversity of the nasopharyngeal, ruminal and vaginal microbiota did not differ between virgin heifers raised from dams exposed to either a low gain (targeted average daily gain of 0.28 kg/d, n = 22) or a moderate gain treatment (0.79 kg/d, n = 23) during the first 84 days of gestation. Only in the vaginal microbiota were there relatively abundant genera that were affected by maternal rate of gain during early gestation. Whilst there was no significant difference in community structure and diversity in any of the three microbiota between pregnant heifers received no VTM (n = 15) and VTM supplemented (n = 17) diets, the VTM supplementation resulted in subtle compositional alterations in the nasopharyngeal and ruminal microbiota. Although the nasopharyngeal, ruminal, and vaginal microbiota were clearly distinct, a total of 41 OTUs, including methanogenic archaea, were identified as core taxa shared across the respiratory, gastrointestinal, and reproductive tracts of both virgin and pregnant heifers.

A mini review on semen sexing in bovine

Sexing of mammalian sperm is a long-standing goal in agricultural industries including cattle breeding. Being able to pre-select the sex of offspring at the time of conception, ranks among the most sought-after reproductive biotechnologies of all time. Sexed semen is commercially offered by most global A.I. companies yielding with 90% accuracy. Fertility of sexed sperm is typically lower compared with conventional sperm. Sexed semen is recommended for breeding only virgin heifers followed by 1st or 2nd lactational cow. The success of sexed semen industry depends upon the sorting speed, accuracy and the fertility of sorted spermatozoa. The use of sexed semen is expected to affect the structure of the dairy industry by creating a greater supply of replacement heifer if reduction in conception rate can be minimized. Today, Flow cytometry is the only successful technique for the sorting of sperm.

Association of genomically enhanced and parent average breeding values with cow performance in Nordic dairy cattle

This study compared the abilities of virgin heifer genomically enhanced breeding values (GEBV) and parent average breeding values (PA) to predict future cow performance. To increase confidence in genomic technology among farmers, a clear demonstration of the relationship between genomic predictions and future phenotypes is needed. We analyzed 12 different traits in first parity, including production, conformation, fertility, and other functional traits. Phenotype data were obtained from national milk recording schemes and breeding values from the Nordic Cattle Genetic Evaluation. Direct genomic breeding values were calculated using genomic BLUP and combined with traditional breeding values, using bivariate blending. The data covered 14,862 Red Dairy Cattle, 17,145 Holstein, and 7,330 Jersey genotyped virgin heifers born between 2013 and 2015 in Denmark, Finland, and Sweden. Phenotypes adjusted for systematic environmental effects were used as measures of cow performance. Two methods were used to compared virgin heifer GEBV and PA regarding their ability to predict future cow performance: (1) correlations between breeding values and adjusted phenotypes, (2) ranking cows into 4 quartiles for their virgin heifer GEBV or PA, and calculating actual cow performance for each quartile. We showed that virgin heifer GEBV predicted cow performance significantly better than PA for the vast majority of analyzed traits. The correlations with adjusted phenotypes were 38 to 136% higher for GEBV than for PA in Red Dairy Cattle, 42 to 194% higher for GEBV in Holstein, and 11 to 78% higher for GEBV in Jersey. The relative change between GEBV bottom and top quartiles compared with that between PA bottom and top quartiles ranged from 9 to 261% for RDC, 42 to 138% for Holstein, and 4 to 90% for Jersey. Hence, farmers in Denmark, Finland, and Sweden can have confidence in using genomic technology on their herds.

Pure-breeding with sexed semen and crossbreeding with semen of double-muscled sires to improve beef production from dairy herds: Factors affecting heifer and cow fertility and the sex ratio

Using sexed semen to produce purebred replacement heifers makes it possible to mate a large proportion of dairy cows to double-muscled sires and to quantitatively and qualitatively improve beef production and increase the income from dairy herds. Net profit first depends on changes in the farm's overall fertility rate. The objective of this study was to analyze the conception rate in herds using a combination of conventional dairy semen (for pure- and crossbreeding), X-sorted dairy semen (to produce purebred replacement heifers), and conventional beef semen (for terminal crossbreeding). Data were obtained from 50,785 inseminations of 15,580 dairy cows (78% Holstein-Friesian, 15% Brown Swiss, 2% Simmental, and 5% crossbreds) from 106 dairy farms (average milk yield 35.1 ± 9.4 kg/d, with 3.76 ± 0.83% fat and 3.32 ± 0.39% protein contents). To account for the main potential confounders, we used separate generalized linear mixed-effects models for cows and virgin heifers. The results showed that the odds ratio of conception improved (1.00 to 1.34) with an increase in the average milk yield of the herd but worsened (1.12 to 0.70) with an increase in the milk yield of individual cows within herd. The summer months showed a strong reduction in the odds ratio of conception in cows (0.56 in July and August) but not in virgin heifers. Multiparous cows had a lower odds ratio of conception (0.85) than primiparous cows (1.00). The order of insemination did not affect the fertility of the cows or heifers, whereas the odds ratio of conception improved with advancing lactation (1.00 to 2.12). The Simmental cows were more fertile than Holstein-Friesians (1.37 vs. 1.00), whereas the fertility of the heifers was not affected by breed. Taking all these possible confounders into account simultaneously, in pure-breeding the odds ratio of conception using sexed semen did not differ from that using conventional dairy semen in cows (0.90 vs. 1.00) or in virgin heifers (0.95 vs. 1.00). However, crossbreeding using conventional beef and dairy semen improved the odds ratio of conception (1.10 and 1.17, respectively) in cows (1.37 using beef semen) and heifers (1.25 using dairy semen). The proportion of newborn heifer calves was ≥90% using sexed dairy semen. The combined use of sexed semen, especially on heifers, to produce purebred replacement females and beef semen to produce terminal crossbred calves was shown to have the potential to increase overall herd fertility, which could be further improved using sexed dairy semen to produce dairy crossbreds instead of purebred replacement heifers.

11 Invivo-derived embryo pregnancy rates at Maddox Dairy from 2008 to 2018

11 Invivo-derived embryo pregnancy rates at Maddox Dairy from 2008 to 2018

Mechanisms linking nutrition and reproduction in postpartum cows

The reproductive physiology of postpartum cows is different from that of heifers because of the combined effects of the past pregnancy and lactation. Neither lactation nor pregnancy has a major effect on postpartum fertility when calving is free from disease and lactation is moderate. Postpartum beef cows in good body condition have conception rates nearly equivalent to those of virgin heifers once their uteri are involuted and they initiate ovarian cycles. However, cows will experience infertility when nutrient requirements for maintenance and lactation exceed nutrient intake (postpartum beef cows) or when nutrients are specifically partitioned toward lactation (postpartum dairy cows). The subsequent loss of body fat that occurs in either case has effects on a variety of reproductive processes and reproduction becomes less efficient. The mechanisms that lead to abnormal reproduction in nutritionally compromised postpartum cattle have been investigated intensively. Much of the effort has focused on the nature of the signal (endocrine or otherwise) that controls pituitary secretion of LH and FSH, the response of the ovary to LH and FSH, and other ovarian effects that are independent of gonadotrophins. Reproductive studies in ruminants have tended toward studies of follicular development and this focus relates back to solving the problem of anoestrus. Less work has been done on the effects of nutrition on the early embryo, the health of which may be predetermined by factors affecting the oocyte within the preovulatory follicle. Few studies have examined the effect of nutrition on uterine function in postpartum cattle. Solutions to postpartum reproduction will probably arise from a variety of approaches that include traditional physiology as well as more modern genomic and proteomic technologies.

Evaluation of Tritrichomonas foetus infection clearance in heifers immunized with a single intravaginal dose of formaldehyde fixed strain B1 cells

Vaccines against Tritrichomonas foetus have been shown to reduce the time of infection after natural or experimental exposure. The object of this study was to assess the protection against T. foetus infection conferred by a single vaginal instillation of formaldehyde fixed T. foetus cells. Aberdeen Angus virgin heifers were randomly allocated to 3 groups of 12 individuals to receive placebo or formaldehyde fixed T. foetus cells prepared following one of two procedures (formalin or freshly prepared solution) and six weeks later they were challenged with 106T. foetus trophozoites. The median time for clearance among control heifers was 93.75 days while in animals immunized with formaldehyde fixed T. foetus it was 45 days. A single vaginal dose of cells fixed with fresh formaldehyde solution gave a rate of decay of infection per unit of time of 2.54 (CI 95% = 1.07;6.01).

Expected value of crossbred dairy cattle artificial insemination breeding strategies in virgin heifers and lactating cows

Research on sexed semen breeding techniques has largely focused on purebred cattle production systems and little work has been done evaluating sexed semen use in crossbred dairy cattle. Complementing a crossbreeding reproduction strategy with sexed semen artificial insemination (AI) has the potential to increase profit potential of sexed semen technology in the short and long run. The objective of this project was to estimate and compare the expected net present value (NPV) of reproduction management programs considering several conventional and sexed semen breeding strategies, cow ages, and breed compositions. While the potential benefits of sexed semen are varied, it is important to note that the focus of this study is on the economic returns to the breeding program. This is not a life-cycle analysis, and therefore, only the sexed semen costs and returns that directly impact the returns to the breeding program are considered. Specifically, a NPV model that incorporates dystocia and stillbirth costs, in addition to improved fertility of crossbred cattle (over purebred cattle), directly into the expected NPV calculation is developed. Increased use of crossbred dairy cattle in the United States make this research particularly timely and relevant. Consistent with previous research, results indicate that pure conventional semen AI and a mixed strategy with one sexed semen AI followed by conventional semen were generally the preferred breeding strategies in terms of expected NPV across all breeds and dam ages evaluated. Similarly, our results also support previous findings that the expected NPV of breeding programs for virgin heifers were consistently higher than lactating cows. In terms of breeds, the crossbreed scenarios generally produced higher expected NPV than scenarios for purebred Holsteins. The most influential factors resulting in these higher returns were the higher cumulative pregnancy rates associated with the crossbreds. Reproductive benefits of crossbred dairy cattle may be able to alleviate some economic pressure currently associated with poor dairy reproduction with AI breeding strategies (especially those incorporating sexed semen). However, it is important to note the likely tradeoffs associated with improvements in fertility (e.g., reduced milk production), the consideration of which is necessary to maximize whole farm profitability.

Inherent inferior quality of follicular fluid in repeat breeder heifers as evidenced by low rates of in vitro production of bovine embryos

The aims of the present study were to determine the effect of follicular fluid obtained from the ovulatory follicle of repeat breeder heifers on in vitro oocyte maturation (Experiment 1), fertilization (Experiment 2) and production of bovine embryos (Experiment 3). Holstein virgin heifers (VH, n = 5) with normal fertility or repeat breeder syndrome (RBH, n = 5) were used in the present study. Follicular fluid of VH and RBH was aspirated from ovulatory follicles and used as maturation medium. Bovine oocytes were aspirated from follicles of slaughterhouse ovaries and randomly allocated in three groups; in Group 1, oocytes cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU/mL hCG plus 0.1 IU/mL rFSH); in Group 2, oocytes cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in Group 3, oocytes cultured in TCM-199 supplemented with 10% filtered follicular fluid of RBH without hormones. The mean (±SEM) percentage of matured oocytes was different between VH and RBH groups (72.2 ± 4.0 vs 56.4 ± 4.6%; P < 0.05, respectively). Further, the mean (±SEM) percentage of normal oocyte fertilization was higher in the VH than the RBH group (49.3 ± 2.1 vs 32.0 ± 4.2; P < 0.05, respectively). The mean percentage of embryos developed to the blastocyst stage was higher in the VH than the RBH group (12.0 ± 1.3 vs 7.0 ± 1.6; respectively; P < 0.05). In conclusion, our findings support our hypothesis that the ovulatory follicle microenvironment of Holstein repeat breeder heifers places their oocytes at a developmental disadvantage compared with Holstein fertile virgin heifers and that this suggest the existence of an inherent inferior quality of the ovulatory follicle microenvironment in repeat breeding Holstein heifers.

Hot topic: 16S rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus

We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.

Cloprostenol sodium and dinoprost tromethamine result in similar artificial insemination pregnancy rates in Bos taurus, Bos indicus, and Bos indicus × Bos taurus cattle synchronized with a Select Synch and CIDR plus timed–artificial insemination protocol

<h2>ABSTRACT</h2> Two experiments were conducted to evaluate breed [Angus (AN), Brahman (BR), and varying percentages of BR × AN breeding] and prostaglandin F_2α (PGF) type [dinoprost tromethamine (dinoprost) vs. cloprostenol sodium (cloprostenol)] in suckled cows (Exp. 1; n=504) and 2-yr-old virgin heifers (Exp. 2; n=309) synchronized with a Select Synch + controlled intravaginal drug-releasing insert and timed-AI protocol (SSCT). On d −7, cows and heifers received gonadotropin-releasing hormone and controlled intravaginal drug-releasing inserts. At controlled intravaginal drug-releasing insert removal (d 0), cows were stratified by BCS, days postpartum, parity, and breed; heifers were stratified by BCS and breed. In both experiments cattle were randomly allotted to receive either dinoprost (25 mg) or cloprostenol (500 μg). Estrus was detected 3 times per day for 72 h after PGF, and AI was conducted by the am/pm rule. At 72 h after PGF, nonresponders were timed artificially inseminated and received gonadotropin-releasing hormone. The AI pregnancy rates were similar (<i>P</i> > 0.05) between PGF treatments in Exp. 1 (51%) and Exp. 2 (46%). In Exp. 1, AN (52%), 1/4 to 3/8 BR (42%), and 1/2 BR (59%) cows had similar AI pregnancy rates, whereas 1/2 BR (59%) had a greater (<i>P</i> < 0.05) AI pregnancy rates compared with cows with ≥3/4 BR (41%). In Exp. 2, heifer breed had no effect on AI pregnancy rates. In conclusion, dinoprost and cloprostenol in a SSCT protocol yielded similar AI pregnancy rates in cows and 2-yr-old virgin heifers across all breed types; however, AI pregnancy rates were influenced by breed type in cows but not 2-yr-old virgin heifers.

Genetic analysis of calving traits by the multi-trait individual animal model

Five alternative models were applied for analysis of dystocia and stillbirth in first and second parities. Models 1 and 2 were included only to estimate the parameters required for model 4, and models 3 and 5 are included only as comparisons to the model 4 estimates. Variance components were estimated by multi-trait REML, including cows with valid calving records for both parities. For the effects of sire of calf on first and second parities, variance components were estimated including only calvings with the same sire of calf for both parities. All heritabilities for the cow effect were quite low, but higher for dystocia than for stillbirth and higher in first parity. The sire-of-calf heritabilities were higher than the cow effect heritabilities, except for stillbirth in parity 2. Unlike the effect of cow correlations, all sire of calf correlations were >0.6, and the correlations for the same trait in parities 1 and 2 were >0.9. Thus, a multi-trait analysis should yield a significant gain in accuracy with respect to the sire of calf effects for bulls not mated to virgin heifers. A multi-trait individual animal model algorithm was developed for joint analysis of dystocia and stillbirth in first and second parities. Relationships matrices were included both for the effects of cow and sire of calf. In addition, random herd-year-season and fixed sex of calf effects were included in the model. Records were preadjusted for calving month and age. A total of 899,223 Israeli Holstein cows with first calvings since 1985 were included in the complete analysis. Approximate reliabilities were computed for both sire of cow and sire of calf effects. Correlations between these reliabilities and reliabilities obtained by direct inversion of the coefficient matrix for a sire of cow-sire of calf model were all close to 0.99. Phenotypic trends for cows born from 1983 through 2007 were economically unfavorable for dystocia and favorable for stillbirth in both parities. Genetic trends were economically unfavorable for both dystocia and stillbirth in first parity. First-parity sire of calf trends were unfavorable for dystocia, but favorable for stillbirth. All environmental trends were nearly zero. Regressions of evaluations of the complete analysis on a model including only calvings before 2011 were all >0.8. All evaluations met the Interbull Method 3 criterion for unbiasedness. Model 4, which computed genetic evaluations for all animals for all 4 traits accounting for all known relationships and correlations among the traits, is recommended for routine genetic evaluation of calving traits.

Short communication: Genetic analysis for fertility traits of heifers and cows from smallholder dairy farms in a tropical environment

The objective of this study was to estimate genetic parameters for various fertility traits on Holstein upgraded dairy heifers and cows in a smallholder system under tropical conditions using data sets from the Thailand national recording scheme. The investigated traits were age at first service (AFS), age at first calving (AFC), days from calving to first service (DTFS), days between first and last service (DFLS), days open (DO), calving interval (CI), number of services per conception (NSPC), and conception at first service (FSC). The data consisted of 68,555, 34,401, and 54,004 records on heifers, primiparous, and multiparous cows, respectively, calving between 1996 and 2011. Gibbs sampling was employed to obtain (co)variance components using both univariate and bivariate analyses with linear and threshold animal models. Virgin heifers had better fertility performance than primiparous and multiparous cows. The reproductive performance in primiparous cows was inferior compared with multiparous cows. Cows with higher Holstein-Friesian blood showed lower reproductive efficiency. Estimated heritabilities for most of the fertility traits were 0.04 or less except for AFS (0.26) and AFC (0.25). The estimated genetic correlations among fertility traits within parity indicated that selection for cows with high conception rate could lead to shortened DO and CI, as well as DTFS. The FSC and NSPC could be used as the best indicators for heifer and cow fertility and could be complemented by other traits, which were genetically considered as different traits such as DTFS and DFLS in terms of a fertility index. This would enable efficient selection for better fertility. Genetic correlations for fertility traits in primiparous and multiparous cows were very high (>0.90), but those between heifers and cows were lower (0.03 to 0.83). The latter results indicated that fertility traits of heifers and cows should be considered as different traits.

Effect of heifer frame score on growth, fertility, and economics.

A non-traditional forage-based protocol was employed to evaluate replacement heifer growth, fertility, and economics between small frame (SF, 3.50; n = 50) and large frame (LF, 5.56; n = 50) heifers using three increasing gain growth phases. Preceding an 85 d growing-breeding period (Phase 3; P3) the heifers were managed as a common group for Phases 1 and 2 (P1 and P2). During P1, heifers grazed common fields of unharvested corn and corn residue (total digestible nutrients [TDN] 56%) with supplemental hay. For P2, heifers grazed early spring crested wheatgrass pasture (CWG; TDN 62%) that was followed by the final P3 drylot growing and breeding period (TDN 68%). Small frame heifers were lighter at the end of P1 in May and at the start of P3 breeding in August (p = 0.0002). Percent of mature body weight (BW) at the end of P1 (209 d) was 48.7% and 46.8%, respectively, for the SF and LF heifers and the percent pubertal was lower for SF than for LF heifers (18.0% vs 40.0%; p = 0.02). At breeding initiation (P3), the percentage of mature BW was 57.8 and 57.2 and the percentage pubertal was 90.0 and 96.0 (p = 0.07) for the SF and LF heifers, respectively; a 5-fold increase for SF heifers. Breeding cycle pregnancy on days 21, 42, and 63, and total percent pregnant did not differ (p>0.10). In drylot, SF heifer dry matter intake (DMI) was 20.1% less (p = 0.001) and feed cost/d was 20.3% lower (p = 0.001), but feed cost/kg of gain did not differ between SF and LF heifers (p = 0.41). Economically important live animal measurements for muscling were measured in May and at the end of the study in October. SF heifers had greater L. dorsi muscle area per unit of BW than LF heifers (p = 0.03). Small frame heifer value was lower at weaning (p = 0.005) and the non-pregnant ending heifer value was lower for SF heifers than for the LF heifers (p = 0.005). However, the total development cost was lower for SF heifers (p = 0.001) and the net cost per pregnant heifer, after accounting for the sale of non-pregnant heifers, was lower for SF heifers (p = 0.004). These data suggest that high breeding efficiency can be attained among March-April born SF and LF virgin heifers when transitioned to a more favorable May-June calving period through the strategic use of grazed and harvested forages resulting in a lower net cost per pregnant SF heifer.

294 EFFECTS OF FOLLICULAR FLUID OBTAINED FROM REPEAT BREEDER DAIRY HEIFER ON MATURATION OF BOVINE OOCYTES IN VITRO

The cause of repeat breeding syndrome is often difficult to explain in dairy heifers with no clinical abnormalities. The aim of the present experiment was to determine the effect of follicular fluid obtained from the preovulatory follicle of repeat breeder heifers on maturation of bovine oocytes in vitro. Holstein virgin heifers either with normal fertility (VH, n = 5) or repeat breeder syndrome (RB, n = 5) were used in the present experiment. The RB heifers had a history of at least 5 unsuccessful consequent artificial breeding. The reason for using such RB heifers was to exclude the possibility of the presence of usual causes of infertility in heifers. Oestrus cycles of all heifers were synchronized using 2 injections of PGF2a 11 days apart. Six to 12 h after oestrus detection, clear follicular fluid samples from the ovulatory follicles were collected transrectally using a long fine-needle covered by a hard plastic tube. Follicular fluid samples were pooled, centrifuged, and frozen until used in the maturation medium. A total of 483 good or excellent quality bovine cumulus-oocytes complexes (COC) were obtained from 2 to 6 mm follicles in diameter from slaughterhouse ovaries and randomly allocated in 3 groups; in group 1 (control, n = 180), oocytes were cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU mL–1 of hCG plus 0.1 IU mL–1 of rFSH); in group 2 (n = 126), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in group 3 (n = 177), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of RB heifers without hormones. All oocytes were cultured for 24 h at 39°C in an atmosphere of 5% CO2 under 90% humidity. At the end of maturation, the degree of cumulus expansion was evaluated and scored under a stereomicroscope. Then, oocytes were mechanically denuded using 3% sodium citrate and repeated pipetting and were fixed in ethanol/acetic acid (3 : 1) for 24 h. The oocytes were subsequently stained with 1% aceto-orcein and evaluated for meiotic resumption. Proportions were statistically analysed using a Chi-squared test (significant at P &lt; 0.05; SPSS program, 11.5). The percentages of fully expanded COC differed among groups (P &lt; 0.001). The maturation rate (MII stage) was 83% (150/180) in oocytes that were cultured in the presence of FCS as the control group. However, a reduction in the maturation rate was observed when oocytes were cultured either in VH follicular fluid (71.4%, 90/126; P &lt; 0.01) or RB follicular fluid (59.3%, 105/177; P &lt; 0.001) compared to the control group. The percentages of matured oocytes were also different between VH and RB follicular fluid (71.4 v. 59.3%; P &lt; 0.01, respectively). In conclusion, the quality of follicular fluid of the preovulatory follicles of repeat breeder heifers is lower than that of the virgin heifers with normal fertility. This may explain the cause of the low fertility in some repeat breeder Holstein heifers.

291 NOVEL OOCYTE SHIPPING AND MATURATION MEDIUM WITHOUT CO2 GAS PHASE IMPROVES IN VITRO MATURATION AND PREGNANCY OUTCOME IN CATTLE

The aim of the present work was to evaluate embryonic development after shipping and maturation of bovine cumulus oocyte complexes (COC) collected by ovum pick up (OPU) in medium (SMM) that does not require CO2 gas for transport and maturation. Two experiments were conducted, experiment 1 stimulated nonlactating Holstein (n = 4), Jersey (n = 2), Angus (n = 4), and Wagyu (n = 2) donors with 6 pFSH injections (Pluset, MOFA Global LLC, Verona, WI, USA) were used. From each donor, some OPU sessions were delivered the same day (~3 h after collection) for IVM in conventional gas bicarbonate-equilibrated medium system (control), while COC from the other sessions were placed in a portable incubator at 38.5°C, delivered the next day allowing 24 h of maturation in SMM (BoviPro, Mofa Global, WI, USA). The COC were fertilized using commercial semen for each breed, and embryos were cultured in BBH7 medium (BoviPro, Mofa Global, WI, USA) at 38.5°C in 5% O2, 5% CO2, and 90% N2 atmosphere. Embryonic development was evaluated in this experiment. For experiment 2, Day 7 fresh Holstein and Jersey embryos (n = 610) from SMM (n = 550) and controls (n = 60) were transferred in synchronized virgin heifers and pregnancies were diagnosed by ultrasonography at d 35. Data were analysed by ANOVA using GLM, percentages were transformed using arcsin square root, and pregnancy rates were analysed by GenMod using SAS statistical software (Cary, NC, USA). Similar COC numbers were recovered for maturation treatments (P &gt; 0.1; Table 1). The COC matured in SMM had higher cleavage and blastocyst rates than the control group (P &lt; 0.01; Table 1), and this resulted in more transferable embryos per OPU session (P &lt; 0.05; Table 1). We did not find breeds effects or interactions for any variable (P &gt; 0.1; Table 1). After ET, SMM had similar pregnancy rates than control (53.8 v. 58.3%; P &gt; 0.1); however, as more blastocysts were produced per OPU session in the SMM condition, more pregnancies were obtained per session (4.3 v. 2.1; P &lt; 0.01). We conclude that COC matured in SMM had greater oocyte competence than control in commercial settings. The SMM resulted in greater embryonic development, similar pregnancy rates, but more transferable embryos and pregnancies per OPU session than the conventional maturation system. Table 1.Least squares means (± SE) of embryonic development of COC matured in SMM or control

Využití metod detekce apoptózy a nekrózy neutrofilních granulocytů krve skotu in vitro

The study was an in vitro analysis of bovine blood neutrophil apoptosis and necrosis based on the detection of their morphological and biochemical features. The experiment was carried out in six clinically healthy Holstein x Bohemian Red Pied crossbred virgin heifers aged 16 to 18 months. The fresh and in vitro cultivated blood were analysed by light and electron microscopy and flow cytometry. In fresh blood, non apoptotic or necrotic neutrophils were found by light microscopy. On the other hand, 3.34% apoptotic and 0.32% necrotic neutrophils were detected by flow cytometry. After four hours of incubation, 3.53% apoptotic and 1.37% necrotic neutrophils were found by light microscopy. In the same conditions, there were assessed 17.54% apoptotic and 0.58% necrotic neutrophils by flow cytometry. Correlation coefficient between light microscopy and flow cytometry in portion of apoptotic neutrophils was 0.625 (P&lt;0.05) and correlation coefficient between light microscopy and flow cytometry in portion of necrotic neutrophils was 0.983 (P&lt;0.05). There was found out that these detection methods (light microscopy and flow cytometry) are right combination for detection of apoptotic and necrotic bovine blood neutrophils.

Expanding the dairy herd in pasture-based systems: The role of sexed semen use in virgin heifers and lactating cows

A model was developed to examine the effects of sexed semen use in virgin heifers and lactating cows on replacement heifer numbers and rate of herd expansion in a seasonal dairy production system. Five separate herds were established according to the type of semen used: conventional frozen-thawed (Conv), sexed fresh semen used in lactating cows for the first 3 wk of the breeding season (SFre1), sexed frozen-thawed semen used in lactating cows for the first 3 wk of the breeding season (SFro1), sexed fresh semen used in lactating cows for the first 6 wk of the breeding season (SFre2), or sexed frozen-thawed semen used in lactating cows for the first 6 wk of the breeding season (SFro2). In the SFro1, SFre1, SFro2, and SFre2 herds, sexed semen was used for the first and second artificial insemination in virgin heifers. Pregnancy rates achieved with sexed fresh and sexed frozen-thawed semen were assumed to be 94 and 75% of those achieved with conventional frozen-thawed semen, respectively. Initial herd size was 100 cows, which was maintained for the first 2 yr of the 15-yr simulation, after which all available replacement heifers were retained to facilitate herd expansion. Two different scenarios of land availability were examined for each of the 5 herds: land available allowed expansion to a maximum herd size of 150 cows (S1), or land available allowed expansion to a maximum herd size of 300 cows (S2). Once maximum herd size was reached, sexed semen use was discontinued and all excess heifer calves were sold at 1 mo old. All capital expenditure associated with expansion was financed with a 15-yr loan. Each of the 10 different options was evaluated in terms of annual farm profit, annual cash flow, and total discounted net profit. The use of fresh sexed semen generated more replacement heifers, leading to faster herd expansion compared with frozen-thawed sexed semen and conventional frozen-thawed semen. Maximum herd size under S1 was reached in yr 5, 5, 4, 5, and 7 for the SFre1, SFro1, SFre2, SFro2, and Conv herds, respectively. Under S2, maximum herd size was reached in yr 8, 10, 7, 9, and 14 for the SFre1, SFro1, SFre2, SFro2, and Conv herds, respectively. Total discounted net profit under S1 for the SFre2 herd was €4,130, €38,869, €47,231, and €52,089 greater compared with the SFre1, SFro1, Conv, and SFro2 herds, respectively. Under S2, discounted net profit for the SFre2 herd was €44,204, €255,524, €280,373, and €325,815 greater compared with the SFre1, SFro1, SFro2, and Conv herds, respectively. The negative effects of the sexed frozen-thawed semen on herd fertility reduced farm profitability and will prevent its widespread use in lactating cows.