Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post estrous onset in cattle.

Introduction: Preovulatory follicle response to the luteinizing hormone (LH) surge leads to metabolic, molecular, and functional changes in the oocyte and somatic follicular cells from the onset of estrus to ovulation. Follicular fluid contains metabolites, miRNAs, proteins, and hormones that are byproducts of follicular metabolism and support cellular processes of oocyte, cumulus, and granulosa constituents. Numerous studies have highlighted the importance of follicular fluid composition to support fertility, but critical gaps exist toward understanding dynamic modifications in the follicular fluid metabolome from estrous onset to ovulation. The hypothesis was that abundance of follicular fluid metabolites is dependent on follicle progression post LH surge and variability in follicular fluid metabolome profiles indicate key processes required for preparation of the follicle and oocyte for optimal fertility. The objective was to generate preovulatory follicular fluid metabolome profiles and discern differences in the metabolome of preovulatory follicular fluid samples collected at onset of estrus, 11 h post estrous onset, and 18 h post estrous onset. Methods: Estrus was synchronized in non-lactating Jersey cows (n=40) and follicular fluid was collected immediately after the first observed standing mount (hr 0) or at approximately h 11 or 18 after the first standing mount. Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry was performed on preovulatory follicular fluid samples (n = 9 collected at hr 0, 9 at h 11, and 10 at h 18) and a multiple linear model was performed to determine if time post estrous onset impacted metabolite abundance. Results: Metabolites influenced by time post estrous onset were tested for enrichment in KEGG pathways. Ninety metabolites were identified in follicular fluid samples. Twenty metabolites differed in abundance among timepoints post estrous onset (p ≤ 0.05). Pathways corresponding to amino acid and energy metabolism were enriched with metabolites impacted by time post estrous onset (FDR ≤ 0.10). Discussion: Results from the current study indicate early response to the LH surge to increase bioavailability of amino acids and metabolites used by the cumulus and granulosa cells for energy production and shuttled into the oocyte to support meiotic maturation. Such metabolites may later be used by the ovulatory follicle for protein production.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Incorporation of a fixed-time artificial insemination protocol results in improved reproductive management and genetics of the beef herd. However, a subset of animals exposed to such protocols will not display estrus prior to insemination. Behavioral estrus is indicative of the preovulatory follicle's physiological maturity and is essential for both the production of an oocyte with optimal developmental competence and preparation of the maternal environment for pregnancy establishment. Animals that do not display estrus prior to insemination and are induced to ovulate a physiologically less advanced follicle have reduced oocyte developmental competence that leads to reduced embryo cleavage rates, embryo quality, and pregnancy rates. This study investigated the impacts of reduced follicle maturity at the initiation of ovulation on the energy production capacity of the oocyte as well as follicular fluid metabolic composition. Results from this study demonstrated that follicle maturity, indicated by increased serum estradiol concentration at the initiation of ovulation, resulted in increased ATP within the oocyte as well as an increased level of metabolites involved in glucose metabolism in the follicular fluid. Increased energy production ability in the oocytes from more mature follicles could contribute to the increased cleavage rates and embryo quality seen in previous studies.

Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS >8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR] < 0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR < 0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.

Extremes in body condition affect fertility and pregnancy outcomes in beef cows. Much research has been done in women and dairy cows to evaluate body condition's effect on oocyte and embryo quality, pregnancy rates, and pregnancy outcomes. However, little work of this type has been done in beef cows and most studies do not focus on the preovulatory time period. The preovulatory time period is an essential time for the oocyte, as final stages of prematuration and the completion of oocyte maturation take place in the peri-ovulatory follicle. The follicular fluid provides the microenvironment for oocyte maturation and exchanges substances with maternal circulation at the blood follicular barrier. Alterations in maternal circulation due to extremes in body condition may pass into the follicular fluid and affect the oocyte during the preovulatory time period. Such conditions may contribute to the reduced fertility seen in beef cows with extreme body condition.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

Approximately 70% of the world's cattle population reside under ambient conditions experiencing some level of heat stress. Heat-stressed cows chronically exposed to elevated ambient temperatures have difficulty getting pregnant. Although the underlying basis for poor fertility during bouts of chronic heat stress remains unclear and is likely because of many different factors, when ambient conditions are sufficient to increase cow body temperature, different ovulatory follicle components are affected (i.e., mural granulosa cells that line the ovulatory follicle, the intrafollicular fluid and or the cumulus-oocyte complex while it matures in preparation for fertilization while resident within). To test this hypothesis, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows. Using steady increases in THI to induce varying levels of elevated body temperature after the luteinizing hormone surge we discovered certain genes in the cumulus cells that may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. We also noted changes in gene expression in granulosa cells that may impact ovulation and corpus luteum formation.

Surgical management and outcome of acquired inguinal hernias in mature bulls: 13 cases (2005-2017).

OBJECTIVE: To describe rates of surgical complications, survival, and return to breeding soundness following herniorrhaphy for bulls with inguinal hernias. ANIMALS: 13 sexually mature bulls with acquired inguinal hernias. PROCEDURES: Medical record databases of 3 veterinary teaching hospitals were searched to identify records of bulls that underwent herniorrhaphy for correction of an inguinal hernia from 2005 to 2017. Information extracted from the medical records included breed, age, duration and side of the hernia, surgical procedure details, postoperative complications, and information regarding subsequent fertility. RESULTS: All 13 bulls had a left inguinal hernia and were anesthetized and underwent herniorrhaphy via an inguinal approach. The left testicle was removed during the surgical procedure in 2 bulls. Nylon or polypropylene mesh secured with size-5 polyester suture was used to facilitate inguinal ring closure in 2 bulls. The inguinal ring was closed with size-5 or size-2 polyester suture in the remaining bulls. Postoperative complications included hernia recurrence (n = 4), excessive scrotal swelling (3), and transient radial nerve paralysis (1). Follow-up information was available for 7 bulls. All 7 bulls had impregnated cows or heifers following surgery, including 1 bull that had hernia recurrence and underwent unilateral castration during the second herniorrhaphy. CONCLUSIONS AND CLINICAL RELEVANCE: Inguinal herniorrhaphy with or without mesh was a safe and effective procedure for inguinal hernia repair in bulls and was associated with a good prognosis for subsequent fertility. However, sparing the ipsilateral testicle during the herniorrhaphy procedure might increase the risk for hernia recurrence.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows.

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Regional selection acting on the OFD1 gene family.

The OFD1 (oral-facial-digital, type 1) gene is implicated in several developmental disorders in humans. The X-linked OFD1 (OFD1X) is conserved in Eutheria. Knowledge about the Y-linked paralog (OFD1Y) is limited. In this study, we identified an OFD1Y on the bovine Y chromosome, which is expressed differentially from the bovine OFD1X. Phylogenetic analysis indicated that: a) the eutherian OFD1X and OFD1Y were derived from the pair of ancestral autosomes during sex chromosome evolution; b) the autosomal OFD1 pseudogenes, present in Catarrhini and Murinae, were derived from retropositions of OFD1X after the divergence of primates and rodents; and c) the presence of OFD1Y in the ampliconic region of the primate Y chromosome is an indication that the expansion of the ampliconic region may initiate from the X-degenerated sequence. In addition, we found that different regions of OFD1/OFD1X/OFD1Y are under differential selection pressures. The C-terminal half of OFD1 is under relaxed selection with an elevated Ka/Ks ratio and clustered positively selected sites, whereas the N-terminal half is under stronger constraints. This study provides some insights into why the OFD1X gene causes OFD1 (male-lethal X-linked dominant) and SGBS2 & JSRDs (X-linked recessive) syndromes in humans, and reveals the origin and evolution of the OFD1 family, which will facilitate further clinical investigation of the OFD1-related syndromes.