Fertility in seasonal-calving pasture-based lactating dairy cows following timed artificial insemination or timed embryo transfer with fresh or frozen in vitro-produced embryos.

The objective was to compare pregnancy per service event (P/S) in lactating dairy cows following timed artificial insemination (AI) or timed embryo transfer (ET) using either fresh or frozen in vitro-produced embryos. Oocytes were collected once per week for up to 9 wk using transvaginal ovum pick-up from elite dairy donors (ET-DAIRY; n = 40; Holstein-Friesian and Jersey) and elite beef donors (ET-ELITE-BEEF; n = 21; Angus). Both ET-DAIRY and ET-ELITE-BEEF donors consisted of heifers and cows. In addition, oocytes were collected from the ovaries of beef heifers of known pedigree following slaughter at a commercial abattoir (ET-COMM-BEEF; n = 119). Following in vitro maturation and fertilization, presumptive zygotes were cultured in vitro to the blastocyst stage. Grade 1 blastocysts were either transferred fresh or frozen for on-farm thawing and direct transfer. A total of 1,106 recipient cows (all lactating, predominantly Holstein-Friesian) located on 16 herdlets were blocked based on parity, calving date, and Economic Breeding Index, and randomly assigned to receive AI (n = 243) or ET (n = 863) after estrous synchronization with a 10-d Progesterone-synch protocol. Cows assigned to ET were further randomized to receive fresh (n = 187) or frozen (n = 178) ET-ELITE-BEEF embryos, fresh (n = 169) or frozen (n = 162) ET-DAIRY embryos, or fresh (n = 80) or frozen (n = 87) ET-COMM-BEEF embryos. Pregnancy was diagnosed using transrectal ultrasound on d 32 to 35 after synchronized ovulation and confirmed on d 62 to 65, at which time fetal sex was determined. Pregnancy per service event at d 32 was not different between AI (48.8%) and ET (48.9%) and did not differ between dairy and beef embryos (50.3% vs. 48.1%, respectively). However, P/S was less on d 32 following transfer of frozen embryos (41.6%) compared with fresh embryos (56.1%). Pregnancy loss between d 32 and 62 was greater for ET (15.1%) compared with AI (4.7%), with greater losses observed for frozen beef (18.5%), fresh beef (17.3%), and frozen dairy (19.2%) compared with fresh dairy (6.0%) embryos. Serum progesterone (P4) concentration on d 7 was associated with P/S at d 32 and 62. Cows in the quartile with the least serum P4 concentrations (quartile 1) had less probability of being pregnant on d 32 (33.4%) compared with cows in the 3 upper quartiles for serum P4 (45.7%, 55.6%, and 61.2% for quartile 2, quartile 3, and quartile 4, respectively). Sex ratio (male:female) at d 62 was skewed toward more male fetuses following ET (61.1:38.9) compared with AI (43.2:56.8) and was consistent with the sex ratio among in vitro blastocysts (61.2:38.8). In conclusion, P/S was similar for AI and ET, although pregnancy loss between d 32 and 62 was greater for ET than for AI.

Effect of delayed timing of artificial insemination with sex-sorted semen on pregnancy per artificial insemination in synchronized dairy heifers managed in a seasonal-calving pasture-based system.

The objective of this study was to evaluate the timing of artificial insemination (AI) with frozen-thawed sex-sorted semen on pregnancy per AI (P/AI) in dairy heifers. A 6-d progesterone Co-Synch protocol was used for ovulation synchronization of dairy heifers, with timed AI (TAI) coincident with (TAI-0) or 8 h (TAI-8) after the second injection of GnRH, corresponding to either 48 h or 56 h after removal of the progesterone-releasing intravaginal device. Pregnancy diagnosis was conducted by transrectal ultrasound scanning of the uterus 34 d after TAI (n = 816 records available for analysis). Generalized linear mixed models were used to examine the effects of treatment on P/AI. Treatment (n = 2), herd (n = 11), and treatment × herd were included as categorical fixed effects. Heifer body weight and Economic Breeding Index values for milk production, fertility, calving performance, beef carcass, cow maintenance, cow management, and health were included as continuous fixed effects. Heifer ID was included as a random effect. Pregnancy per AI was greater for TAI-8 heifers (59%) compared with TAI-0 heifers (50%). Pregnancy per AI ranged from 38% to 75% between herds but there was no treatment × herd interaction. The fertility subindex (positive) and the cow management subindex (negative) were the only continuous animal variables associated with P/AI. Delaying the timing of AI with frozen-thawed sex-sorted semen by 8 h in dairy heifers enrolled on a 6-d progesterone Co-Synch protocol improved P/AI.

Associations between the postpartum uterine and vaginal microbiota and the subsequent development of purulent vaginal discharge vary with dairy cow breed and parity.

The objective of this study was to characterize the species composition and functional potential of the vaginal and uterine microbiota at 1 wk postpartum in dairy cows diagnosed with or without purulent vaginal discharge (PVD) at 3 wk postpartum. The hypothesis was that differences in the vaginal and uterine microbiota between cows diagnosed with (PVD+) or without (PVD-) PVD were dependent on parity and breed. Cytobrush samples of the vagina and uterus were collected at 1 wk postpartum from 36 Holstein-Friesian (7 primiparous and 29 multiparous) and 29 Jersey (10 primiparous and 19 multiparous) cows. Microbial DNA was isolated from each sample and processed for shotgun metagenomic sequencing. The odds of multiparous cows being diagnosed as PVD+ was less compared with primiparous cows (OR = 0.21). Neither the α-diversity nor ß-diversity of the uterine and vaginal microbiota were associated with PVD but the ß-diversity was different between breeds and between parities. In the vagina of primiparous cows, differences in the microbiota of PVD- and PVD+ cows were minor, but the microbiota of multiparous PVD+ cows had greater relative abundance of Fusobacterium necrophorum, Trueperella pyogenes, Porphyromonas levii, and greater functional potential for amino acid and protein synthesis, energy metabolism, and growth compared with PVD- cows. The uterus of primiparous PVD+ cows had lesser relative abundance of Bacteroides heparinolyticus compared with PVD- cows. In the uterine microbiota, differences included greater functional potential for cellulose biosynthesis and fucose catabolism in multiparous PVD+ cows compared with PVD- cows. In the uterine microbiota of primiparous PVD+ cows, the functional potential for gram-negative cell wall synthesis and for negative regulation of tumor necrosis factor signaling was lesser compared with multiparous PVD+ cows. In the vagina of Holstein-Friesian PVD+ cows, the relative abundance of Caviibacter abscessus was greater whereas in the vagina of Jersey PVD+ cows the relative abundance of Catenibacterium mitsuokai, Finegoldia magna, Klebsiella variicola, and Streptococcus anginosus was greater compared with PVD- cows. In the uterine microbiota of Holstein-Friesian cows, the functional potential for spermidine biosynthesis was reduced compared with PVD- cows. In summary, differences in the species composition and functional potential of the vaginal and uterine microbiota between PVD- and PVD+ cows were dependent on parity and breed. The findings suggest that alternative strategies may be required to treat PVD for different parities and breeds of dairy cow.

Erratum to "Reproductive performance of early- and late-calving dairy cows artificially inseminated after ovulation synchronization and estrous resynchronization or artificially inseminated after observed estrus" (JDS Commun. 2:80-85).

[This corrects the article DOI: 10.3168/jdsc.2020-0035.].

TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation.

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). Combinations of PAMPs can trigger synergistic immune responses, but the underlying molecular mechanisms of synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with monophosphoryl lipid A (MPLA) and CpG as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for dual adjuvant immune responses. PLP-based co-delivery of MPLA and CpG to GM-CSF-driven mouse bone marrow-derived antigen-presenting cells (BM-APCs) elicited synergistic interferon-ß (IFN-ß) and interleukin-12p70 (IL-12p70) responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that MyD88 and interferon regulatory factor 5 (IRF5) were necessary for IFN-ß and IL-12p70 production, while TRIF signaling was required for the synergistic response. Both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling drove the synergy. These results identify the key mechanisms of synergistic Toll-like receptor 4 (TLR4)-TLR9 co-signaling in mouse BM-APCs and underscore the critical role of signaling kinetics and biophysical properties on the integrated response to combination adjuvants.

Reproductive performance of early- and late-calving dairy cows artificially inseminated after ovulation synchronization and estrous resynchronization or artificially inseminated after observed estrus.

Within seasonal dairy systems, cows that calve late in the calving season are less likely to become pregnant and maintain a yearly calving interval. Very few studies have examined effective strategies for reproductive management of these late-calving cows. The objectives were to evaluate the reproductive performance of early- and late-calving dairy cows that were either inseminated after observed estrus (control) or enrolled in a timed AI and resynchronization protocol [progesterone (P4) Ovsynch Resynch)]. Early-calving cows calved during the first week of the calving season, whereas late-calving cows calved after 6 wk but were at least 10 d in milk at study commencement. Three dairy herds participated in the study with 391 cows total. Within each calving group, cows were randomly assigned to P4 Ovsynch Resynch or control (no treatment) in a 2 × 2 experimental design. Artificial insemination continued for 6 wk after mating start date (MSD) and was followed by 6 wk of natural service. The interval from MSD to AI was shorter (11.7 vs. 14.7 d) and the 3-wk pregnancy rate (49.5 vs. 21.2%) and the 6-wk pregnancy rate (60.8 vs. 42.4%) were greater in the early-calving compared with the late-calving control cows. By design, synchronized cows received timed AI on MSD and were not included in the statistical analysis of submission rate and interval from MSD to AI. The proportion of cows that received a second AI was not increased by the progesterone-based resynchronization strategy but was greater in early-calving compared with late-calving cows. The 12-wk pregnancy rate was greater (64.5 vs. 45.0%) in the early-calving synchronized cows compared with the late-calving synchronized cows. The interval from MSD to pregnancy was 6 and 12.6 d shorter for synchronized compared with control cows in the early- and late-calving groups, respectively. The results demonstrated reduced reproductive performance of late-calving cows compared with early-calving cows. Nonetheless, a major improvement to reproductive performance was achieved by targeting late-calving cows with a synchronization program, even when cows were only 20 to 50 DIM at first AI. Resynchronization of estrus with a progesterone device only, however, was not sufficient to increase the proportion of nonpregnant cows that received a second AI.

Monitoring estrous activity in pasture-based dairy cows.

Correctly identifying cows in estrus and inseminating them at the optimal time are critical components of reproductive management. Technologies for detecting cows in estrus have developed from tail paint in the 1970's to automated activity monitors and mount detectors in recent decades. The objectives of this study were to identify animal characteristics associated with estrous behaviour, measured using a mount detector (FlashMate; Farmshed Labs) and an accelerometer-based activity monitor (Moomonitor, Dairymaster), to examine the relationship between estrous behaviour measured by both devices, and to examine the characteristics associated with pregnancy per AI. Four hundred and sixty eight lactating dairy cows managed on three research farms were enrolled and data were available from 465 cows and 369 cows with Moomonitor and FlashMate data, respectively. Of 234 cows that provided both Moomonitor and FlashMate data, the mean (±SEM) onset of device activation occurred 1.1 (±0.4) h earlier with the Moomonitor compared with the FlashMate. The mean (±SD) duration of Moomonitor activity and FlashMate activity was 17.2 ± 6.1 h and 8.7 ± 5.8 h, respectively. The duration of Moomonitor activity and FlashMate activity was negatively associated with total milk yield during the first five weeks of lactation. The duration of FlashMate activity, but not the duration of Moomonitor activity was positively associated with days in milk. Pregnancy per AI was positively associated with BCS, days in milk, the duration of FlashMate activity and the interval from the onset of Moomonitor and FlashMate activity to AI. Inseminating cows ≤2 h after FlashMate activation or ≤4 h after Moomonitor activation was associated with reduced odds of pregnancy compared with later timing of AI. Overall, 55% of cows received mounts for ≤8 h, highlighting the need for ≥3 periods of estrous observation daily or the use of estrous detection aids that continuously monitor cows. Finally, the study reiterated the importance of maximising body condition score and days in milk at breeding to increase behavioural expression of estrus and pregnancy per AI.

Evaluation of delayed timing of artificial insemination with sex-sorted sperm on pregnancy per artificial insemination in seasonal-calving, pasture-based lactating dairy cows.

The objective was to use ovulation synchronization with timed artificial insemination (TAI) to evaluate the effect of timing of artificial insemination (AI) with frozen sex-sorted sperm on fertility performance in pasture-based compact calving herds. Ejaculates from 3 Holstein-Friesian bulls were split and processed to provide frozen sex-sorted sperm (SS) at 4 × 106 sperm per straw, and frozen conventional sperm at 15 × 106 sperm per straw (CONV). A modified Progesterone-Ovsynch protocol was used for estrous synchronization, with TAI occurring 16 h after the second GnRH injection for cows assigned to CONV, and either 16 h (SS-16) or 22 h (SS-22) for cows assigned to SS. Pregnancy diagnosis was conducted by transrectal ultrasound scanning of the uterus 35 to 40 d after TAI (n = 2,175 records available for analysis). Generalized linear mixed models were used to examine the effects of treatment on pregnancy per artificial insemination (P/AI). Fixed effects included treatment (n = 3), bull (n = 3), treatment by bull interaction, parity (n = 4), days-in-milk category (n = 3), and treatment by days-in-milk category, with herd (n = 24) included as a random effect. Pregnancy per AI was greater for CONV compared with both SS-16 and SS-22 (61.1%, 49.0%, and 51.3%, respectively), and the SS treatments did not differ from each other (relative P/AI for SS-16 and SS-22 vs. CONV were 80.2% and 84.0%, respectively). There were significant bull and treatment by bull interaction effects. Additional analysis was undertaken using a model that included herd as a fixed effect. This analysis identified marked herd-to-herd variation (within-herd relative P/AI for the combined SS treatments vs. CONV ranged from 48-121%). The tertile of herds with the best performance achieved a mean relative P/AI of 100% (range = 91-121%), indicating that P/AI equivalent to CONV is achievable with SS. Conversely, the tertile of herds with the poorest performance achieved a mean relative P/AI of 67% (range = 48-77%). We found that SS resulted in poorer overall P/AI compared with CONV sperm regardless of timing of AI. Marked variation existed between herds; however, one-third of herds achieved P/AI results equal to CONV. Identification of factors responsible for the large herd-to-herd variation in P/AI with SS, and development of strategies to reduce this variation, warrant further research.

Ovarian function and the establishment and maintenance of pregnancy in dairy cows with and without evidence of postpartum uterine disease.

Uterine disease early postpartum reduces fertility during the breeding period. One potential mechanism involves the reduced functional capacity of the uterus to support pregnancy. A second potential mechanism involves damage to ovarian follicles associated with systemic inflammation. We categorized lactating Holstein cows into healthy (n = 63) and diseased (n = 39) uterus groups based on the percentage of polymorphonuclear neutrophils in the uterine lumen during the second and third month postpartum and evaluated the functionality of their ovaries and their capacity to establish and maintain pregnancy. Cows were enrolled in a timed artificial insemination protocol (Presynch Ovsynch) so that the first artificial insemination was approximately 75 d postpartum. Ovarian follicles and corpora lutea were counted and measured using transrectal ultrasound, ovulatory responses were assessed, and luteal phase progesterone concentrations were measured. Pregnancy was detected on d 18, 20, 22, 25, 32, and 45 through chemical (d 18 to 25) or ultrasonographic methods (d 32 and 45). The percentage of cows ovulating during the Presynch period; the number, diameter, and ovulatory capacity of follicles during the Ovsynch period; and plasma progesterone concentrations following ovulation were similar for healthy and diseased cows. The initial period of pregnancy establishment (d 18 to 22) appeared to be unaffected by disease because a similar percentage of healthy and diseased cows were pregnant during this period. Embryonic loss occurred in both healthy and diseased cows after d 22. Based on a relatively small number of pregnancies (n = 30 healthy and n = 17 diseased), the cumulative embryonic loss after d 22 was greater in diseased compared with healthy cows. In short, uterine disease as defined in this study did not affect cyclicity, ovarian follicular growth, or plasma progesterone concentrations. Percentages of healthy and diseased cows that were pregnant were similar from d 18 to 22 after artificial insemination. Greater embryonic loss was observed after d 22 in diseased compared with healthy cows, but this observation was based on a small number of pregnancies and should be studied further in larger trials with greater statistical power.

Growth of the conceptus from days 33 to 45 of pregnancy is similar for heifers and lactating cows and not associated with circulating glucose, insulin, IGF1 or progesterone concentrations.

Less than optimal circulating progesterone as well as glucose, insulin, and IGF1 concentrations in lactating cows may contribute to embryonic loss if placental development is dependent on concentrations of these hormones. There was comparison of fetal growth in lactating dairy cows with a contemporary group of nulliparous heifers (negative control for lactation). There was also assessment of the association between conceptus development and circulating hormone and metabolite concentrations and the response to an intravenous glucose administration. The embryo and amniotic vesicle were measured every 2-3 days during a 2-week period (days 33-45 of pregnancy). Blood samples were collected at the time of ultrasonography and analyzed for glucose, insulin, IGF1, and progesterone. There was also an intravenous glucose administration. Lactating cows had lesser blood concentrations of glucose, progesterone, and IGF1 during pregnancy. With respect to glucose administration, cows had lesser insulin and glucose than heifers at time 0 but heifers released three-fold more insulin than cows in response to glucose infusion. Cows had slightly larger fetuses when compared with heifers. When included in the model for embryo or amniotic vesicle length or area, concentrations of circulating glucose, insulin, IGF1, or progesterone did not explain a large amount of variation in values for these variables (P > 0.10). The conclusion was that cows differed from heifers for fetal growth but concentrations of circulating metabolites were not associated with size of the fetus. There was no evidence for an association between the hormonal or metabolic milieu and conceptus development.

Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development.

The hypothesis of this study was that cows with good genetic merit for fertility traits (Fert+) would produce oocytes and embryos of greater quality than cows with poor genetic merit for fertility traits (Fert-) and that mRNA expression of candidate genes would reflect the observed differences in quality. The aim of the study, therefore, was to determine the effect of genetic merit for fertility traits on morphological classification and mRNA abundance of key genes in immature oocytes and cumulus cells following ovum pick-up and in embryos following superovulation, artificial insemination (AI), and uterine flushing. In experiment 1, 17 Fert+ and 11 Fert- cows, ranging from 54 to 84 d in milk, were submitted to ovum pick-up on 4 occasions during a 2-wk period. Recovered cumulus-oocyte complexes (COC) were morphologically graded. Oocytes and cumulus cells were separated, and mRNA abundance of genes associated with oocyte developmental competence was measured. There was no effect of genotype on the distribution of COC grades or on the mRNA abundance of the candidate genes in grade 1 COC. In experiment 2, 20 Fert+ and 19 Fert- cows, ranging from 71 to 189 d in milk, were submitted to superovulation and AI. The uteri of cows that responded to the superovulation protocol (17 Fert+ and 16 Fert- cows) were nonsurgically flushed 7 d postovulation. Recovered embryos were morphologically graded, and mRNA abundance of genes associated with embryo development was measured in grade 1 blastocysts. The response to the superovulation protocol was assessed by counting the number of codominant follicles on the day of AI, which was similar for both genotypes (22.0 ± 9.7 and 19.8 ± 8.2 for Fert+ and Fert- cows, respectively). There was no effect of genotype on the proportion of transferable embryos recovered or on the mRNA abundance of the candidate genes tested in the grade 1 blastocysts. Of the total embryos classified as blastocysts, however, the Fert+ cows tended to have a greater proportion of grade 1 blastocysts compared with Fert- cows (90% vs. 64%, respectively). In conclusion, genetic merit for fertility traits had a no effect on mRNA abundance of the candidate genes that were examined in immature oocytes and cumulus cells and in embryos recovered after superovulation. The observed differences in morphological blastocyst quality following superovulation would suggest that the superior reproductive performance of Fert+ cows could arise during the later stages of embryo development from d 7 until maternal recognition of pregnancy.

Genome-wide study to detect single nucleotide polymorphisms associated with visceral and subcutaneous fat deposition in Holstein dairy cows.

Excessive abdominal fat might be associated with more severe metabolic disorders in Holstein cows. Our hypothesis was that there are genetic differences between cows with low and high abdominal fat deposition and a normal cover of subcutaneous adipose tissue. The objective of this study was to assess the genetic basis for variation in visceral adiposity in US Holstein cows. The study included adult Holstein cows sampled from a slaughterhouse (Green Bay, WI, USA) during September 2016. Only animals with a body condition score between 2.75 and 3.25 were considered. The extent of omental fat at the level of the insertion of the lesser omentum over the pylorus area was assessed. A group of 100 Holstein cows with an omental fold <5 mm in thickness and minimum fat deposition throughout the entire omentum, and the second group of 100 cows with an omental fold ⩾20 mm in thickness and with a marked fat deposition observed throughout the entire omentum were sampled. A small piece of muscle from the neck was collected from each cow into a sterile container for DNA extraction. Samples were submitted to a commercial laboratory for interrogation of genome-wide genomic variation using the Illumina BovineHD Beadchip. Genome-Wide association analysis was performed to test potential associations between fat deposition and genomic variation. A univariate mixed linear model analysis was performed using genome-wide efficient mixed model association to identify single nucleotide polymorphisms (SNPs) significantly associated with variation in a visceral fat deposition. The chip heritability was 0.686 and the estimated additive genetic and residual variance components were 0.427 and 0.074, respectively. In total, 11 SNPs defining four quantitative trait locus (QTL) regions were found to be significantly associated with visceral fat deposition (P<0.00001). Among them, two of the QTL were detected with four and five significantly associated SNPs, respectively; whereas, the QTLs detected on BTA12 and BTA19 were each detected with only one significantly associated SNP. No enriched gene ontology terms were found within the gene networks harboring these genes when supplied to DAVID using either the Bos taurus or human gene ontology databases. We conclude that excessive omental fat in Holstein cows with similar body condition scores is not caused by a single Mendelian locus and that the trait appears to be at least moderately heritable; consequently, selection to reduce excessive omental fat is potentially possible, but would require the generation of predicted transmitting abilities from larger and random samples of Holstein cattle.

Technical note: Evaluation of fine needle aspiration cytology for the diagnosis of fatty liver in dairy cattle.

Fatty liver is a common condition affecting dairy cattle during the periparturient period, characterized by a pathological accumulation of triglycerides (TG) in the hepatocytes. The objective of this study was to evaluate the diagnostic potential of fine needle aspiration cytology in fresh liver specimens using liver TG concentrations as a gold standard. Fifty-seven liver samples from Holstein cows were collected during processing at a slaughterhouse. Tissue and fine needle aspirate samples were obtained from the parietal upper portion of the caudate lobe. Two samples of liver tissue were collected with a 16 gauge × 15 cm biopsy needle for histological and TG concentration assessment. A third sample was collected for cytology using an 18 gauge × 5.08 cm needle. The contents of the needle were transferred to a glass slide, spread, and air-dried. Liver samples were assayed by colorimetry/fluorimetry to determine TG concentrations. Concentrations of TG <2% were considered normal. Histological and cytological evaluations were conducted by 2 different pathologists blind to the visual classification. Sensitivity (Se) and specificity (Sp) were calculated. Cytology had a Se and Sp of 73 and 85%, respectively. Histopathology had a Se and Sp of 45.9 and 100%, respectively. The likelihood of having higher scores for histopathology and cytology increased as a function of liver TG content (mg/g).

A 100-Year Review: Reproductive technologies in dairy science.

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks. Improved semen extenders and the replacement of glass ampules with plastic semen straws followed. Some of the most noteworthy developments in the 1970s included the initial successes with in vitro culture of embryos, calves born after chromosomal sexing as embryos, embryo splitting resulting in the birth of twins, and development of computer-assisted semen analysis. The 1980s brought flow cytometric separation of X- and Y-bearing sperm, in vitro fertilization leading to the birth of live calves, clones produced by nuclear transfer from embryonic cells, and ovum pick-up via ultrasound-guided follicular aspiration. The 20th century ended with the birth of calves produced from AI with sexed semen, sheep and cattle clones produced by nuclear transfer from adult somatic cell nuclei, and the birth of transgenic cloned calves. The 21st century has seen the introduction of perhaps the most powerful biotechnology since the development of artificial insemination and cryopreservation. Quick, inexpensive genomic analysis via the use of single nucleotide polymorphism genotyping chips is revolutionizing the cattle breeding industry. Now, with the introduction of genome editing technology, the changes are becoming almost too rapid to fully digest.

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.

Follicular fluid and serum metabolites in Holstein cows are predictive of genetic merit for fertility.

The aims of the present study were to: (1) characterise the metabolome of follicular fluid and serum in dairy cows with similar genetic merit for milk production but with extremes of good (Fert+) or poor (Fert-) genetic merit for fertility; and (2) identify potential biomarkers of dairy cow fertility. Follicular fluid from the first wave dominant follicle and serum were collected on Day 7 of the oestrous cycle. The most pronounced effect of genotype was noted in the serum, where the abundance of total polyunsaturated fatty acids and n-6 polyunsaturated fatty acids was greater in Fert+ cows, and the abundance of total saturated fatty acids was greater in Fert- cows. The abundance of nine fatty acids (arachidic acid, heneicosanoic acid, myristic acid, behenic acid, myristoleic acid, heptadecenoic acid, cis-11-eicosanoic acid, nervonic acid and γ-linolenic acid) in follicular fluid was affected by genotype. Concentrations of cysteine, leucine, ornithine, proline and tyrosine in follicular fluid, and asparagine, creatinine, cysteine, methionine, proline and valine in serum, were also affected by genotype. Receiver operating characteristic curve analysis indicated that the follicular fluid and serum fatty acids and follicular fluid amino acids that were significantly affected by genotype were highly predictive of fertility genotype.

Technical note: Validation of a chemical pregnancy test in dairy cows that uses whole blood, shortened incubation times, and visual readout.

Chemical pregnancy testing is an alternative to traditional methods of pregnancy diagnosis (either manual palpation or ultrasound) in postpartum dairy cows and heifers. The objective was to validate a chemical pregnancy test that confers the advantages of using whole blood, rapid incubation times, and visual readout. Blood and milk samples were collected from Holstein dairy cows [n=320; 162±62 (mean ± SD) d in milk] on a confinement farm in northeast Missouri at 28 d after artificial insemination (AI). The samples were assayed for pregnancy-associated glycoproteins (PAG) by using a rapid visual test as well as traditional plasma- and milk-based tests. Transrectal ultrasonography diagnosis for pregnancy at 35 to 38 d after AI was the reference (gold) standard for all PAG tests. One hundred fifty-nine cows were diagnosed as pregnant by the reference standard (pregnancies per AI=49.7%). The tests were ELISA and either optical density (OD; measured with a microtiter plate reader; plasma, milk, and rapid visual tests) or visual readout (rapid visual test) were used to diagnose pregnancy. When OD was used, the percentage of pregnant cows classified correctly (sensitivity) for the plasma, milk, and rapid visual tests were 97±1, 96±2, and 95±1% (±SE), respectively. The sensitivity of the rapid visual test when assessed visually was 98±1%. The specificity (proportion of nonpregnant cows classified correctly) for the plasma, milk, and rapid visual was 94±2%, 94±2%, and 93±2% when an OD was used. When read visually, the specificity of the rapid visual test was lesser (85±3%) because some cows with faint visual signals yielded false positive diagnosis. The overall accuracy (proportion of pregnant and nonpregnant cows diagnosed correctly) was similar for all tests (plasma, milk, rapid visual OD, and rapid visual; 96±1, 95±1, 94±1, and 92±2%, respectively). In a second experiment, lactating Holstein cows (n=291) from 4 commercial confinement dairy farms in western Kentucky were tested 25 to 95 d after AI using the rapid visual test. The OD of the rapid visual test followed the known profile for PAG in circulation during the first trimester of pregnancy. The conclusion is that the rapid visual test has equal sensitivity and accuracy as existing PAG tests. A slightly lower specificity was found when the rapid visual test was read visually.

Growth of the conceptus from day 33 to 45 of pregnancy is minimally associated with concurrent hormonal or metabolic status in postpartum dairy cows.

A hypothetical explanation for pregnancy loss in postpartum dairy cows is that the metabolic environment of the cow inhibits the growth of the conceptus and places the pregnancy at risk for loss. The objective of the current study, therefore, was to model the association between cow-level metabolic indicators and conceptus growth during early pregnancy (day 33-45 after AI) and to determine if an association (if present) is large enough to cause pregnancy loss. Metabolic indicators included milk production, changes in body weight and body condition score, parity, and concentrations of circulating hormones and metabolites (glucose, non-esterified fatty acids, growth hormone, IGF1, progesterone, and pregnancy-associated glycoproteins). One-hundred cows were enrolled. Cows that became pregnant with single conceptus pregnancies (n=53) weighed more (P<0.007) and had fewer uterine polymorphonuclear neutrophils (uterine health indicator; P<0.051) compared with cows that failed to become pregnant. The embryo and amniotic vesicle were measured by using ultrasound on day 33, 35, 38, 40, 42, and 45 of pregnancy. Most of the cow-level indicators that were included in the model of conceptus growth failed to achieve statistical significance. Day of pregnancy had the largest effect on conceptus growth (size and cross-sectional area of the embryo and amniotic vesicle; P<0.001). There were effects of sex of fetus (male fetuses larger than female), insulin (negative association), and body weigh change (positive association) on embryo length and cross-sectional area but these effects were small when compared with the range in conceptus length or area that we observed. The conclusion was that the capacity of the cow to become pregnant was associated with body weight and uterine health but we failed to find a large association with metabolic status on conceptus growth from day 33 to 45 of pregnancy in lactating dairy cows.

Genetic parameters of dairy cow energy intake and body energy status predicted using mid-infrared spectrometry of milk.

Energy balance (EB) and energy intake (EI) are heritable traits of economic importance. Despite this, neither trait is explicitly included in national dairy cow breeding goals due to a lack of routinely available data from which to compute reliable breeding values. Mid-infrared (MIR) spectrometry, which is performed during routine milk recording, is an accurate predictor of both EB and EI. The objective of this study was to estimate genetic parameters of EB and EI predicted using MIR spectrometry. Measured EI and EB were available for 1,102 Irish Holstein-Friesian cows based on actual feed intake and energy sink data. A subset of these data (1,270 test-day records) was used to develop equations to predict EI, EB, and daily change in body condition score (ΔBCS) and body weight (ΔBW) using the MIR spectrum with or without milk yield also as a predictor variable. Accuracy of cross-validation of the prediction equations was 0.75, 0.73, 0.77, and 0.70 for EI, EB, ΔBCS, and ΔBW, respectively. Prediction equations were applied to additional spectral data, yielding up to 94,653 records of MIR-predicted EI, EB, ΔBCS, and ΔBW available for variance component estimation. Variance components were estimated using repeatability animal linear mixed models. Heritabilities of MIR-predicted EI, EB, ΔBCS, and ΔBW were 0.20, 0.10, 0.07, and 0.06, respectively; heritability estimates of the respective measured traits were 0.35, 0.16, 0.07, and 0.08, respectively. The genetic correlation between measured and MIR-predicted EI was 0.84 and between measured and MIR-predicted EB was 0.54, indicating that selection based on MIR-predicted EI or EB would improve true EI or EB. Genetic and phenotypic associations between EI and both the milk production and body-change traits were generally in agreement, regardless of whether measured EI or MIR-predicted EI was considered. Higher-yielding animals of higher body weight had greater EI. Predicted EB was negatively genetically correlated with milk yield (genetic correlation=-0.29) and positively genetically correlated with both milk fat and protein percent (genetic correlation=0.17 and 0.16, respectively). Least squares means phenotypic EI of 198 animals stratified as low, average, and high estimated breeding values for MIR-predicted EI (animal phenotypes were not included in the genetic evaluation) were 154.3, 156.0, and 163.3 MJ/d, corroborating that selection on MIR-predicted EI will, on average, result in differences in phenotypic true EI.