Modulation of expression of estrus, steroidogenesis and embryo development following peri-artificial insemination nutrient restriction in beef heifers.

Nutritional changes immediately after insemination cause increased embryonic mortality, but the mechanisms controlling this are not well known. Our objective was to evaluate the impact of nutritional change on estrus expression, steroid concentrations, peripheral and uterine luminal fluid metabolites, and embryo quality in beef heifers. Heifers (n = 139) were assigned to one of two pre-artificial insemination (AI) dietary treatments: LOW (≤ 90% NEm) or HIGH (≥ 139% NEm). Heifers were on treatment for 33-36 days before AI (d0) when half of the heifers in each treatment were randomly reassigned to generate four treatments; HIGH-HIGH, HIGH-LOW, LOW-HIGH, and LOW-LOW. Heifers remained on treatments until embryo collection (d 6-8). Negative energy balance was achieved among LOW heifers as demonstrated by body weight loss and increased NEFA concentrations (P < 0.05). Pre-AI treatment influenced expression of estrus (P = 0.05; HIGH 80.4 ± 4.0% vs. LOW 69.4 ± 4.2%). Estradiol concentrations and interval to estrus were not affected by treatment (P > 0.55); however, progesterone concentrations were reduced among LOW compared to HIGH (3.57 ± 0.27, 4.64 ± 0.26 ng/mL, respectively; P = 0.004), and heifers maintained on the HIGH pre-AI diet had consistently greater concentrations of progesterone from d 0 to d 8 (P = 0.014). Pre-AI treatment influenced embryo stage (P = 0.05; HIGH 3.61 ± 0.32 vs. LOW 2.72 ± 0.30). Post-AI treatment affected embryo grade (P = 0.02; HIGH 1.78 ± 0.23 vs. LOW 2.64 ± 0.27). In summary, pre-AI nutrient restriction caused decreased expression of estrus, reduced progesterone concentrations after AI, and negatively impacted embryo development, while post-AI restriction hindered embryo quality.

Proteomics dataset of epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm from Angus bulls.

Peptides and proteins were identified by liquid chromatography with tandem mass spectrometry analysis (LCMS-MS) on an Orbitrap Velos mass spectrometer to further understand biological mechanisms that regulate increased longevity in epididymis compared to ejaculated sperm. Semen from sexually mature bulls were collected and then bulls were slaughtered to collect epididymal samples from the cauda epididymis. All samples were centrifuged to separate spermatozoa from fluids. A high ionic solution was used to remove surface proteins from spermatozoa. Four unique samples were generated: (1) epididymal fluid, (2) seminal plasma (ejaculated fluid), and proteins stripped from (3) epididymal sperm, and (4) ejaculated sperm. Samples were analyzed by LCMS-MS, and data were interpreted with Protein Pilot 5. False discovery rate (FDR) was set at 1%. Unique proteins (n = 458) were identified in ejaculated samples, 178 proteins in seminal plasma and 298 proteins stripped from ejaculated sperm. In epididymal samples, 311 proteins were identified in the fluid, and 334 were identified among proteins stripped from epididymal sperm. This dataset can be useful for further understand of biological mechanisms that control sperm longevity. This dataset is related to the article 'Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity' by (Zoca et al., 2022).

Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity.

Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 °C. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 °C. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR <0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm's increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH.

Effect of progesterone supplementation in a resynchronization protocol on follicular dynamics and pregnancy success.

The objective of this study was to evaluate the necessity of a controlled internal drug releasing device (CIDR) in a fixed-time AI resynchronization protocol as well as to compare a commercially available blood pregnancy test with transrectal ultrasonography for Day 28 pregnancy detection. Over a two-year period, beef cows and heifers from twelve herds were inseminated using the 7-day CO-Synch + CIDR protocol. On Day 21 following the first insemination, the protocol was repeated, with animals receiving either a CIDR or no CIDR. Pregnancy status (AI1) was determined on Day 28 by both transrectal ultrasonography and the IDEXX Rapid Visual Pregnancy Test. Non-pregnant animals by both methods (CIDR: n = 190 cows, n = 228 heifers; no CIDR: n = 185 cows, n = 223 heifers) received an injection of Prostaglandin F2alpha and were inseminated at the appropriate time or bred following detection of estrus. Corpora lutea (CL) number and largest follicle diameter were recorded on a subset of non-pregnant animals (CIDR: n = 66 cows, n = 46 heifers; no CIDR: n = 76 cows, n = 41 heifers) at time of pregnancy diagnosis on Day 28. Final pregnancy status was determined a minimum of 31 days following the second AI (AI2). The GLIMMIX procedure of SAS was utilized for estrus and pregnancy data; while the MIXED procedure was utilized for analyses of CL number and largest follicle diameter. There was no effect (P ≥ 0.55) of treatment on AI1 pregnancy, AI2 pregnancy, or overall pregnancy rates. The presence of a CIDR during the resynchronization increased (P < 0.001) estrus expression prior to AI2. There was an effect of treatment by age on AI2 pregnancy (P < 0.01); heifers that received a CIDR had greater AI2 pregnancy rates than heifers that did not receive a CIDR (P = 0.04), but there was no difference between cows with and without a CIDR. Treatment had no effect (P > 0.10) on embryonic loss (between the first and second pregnancy diagnosis), CL number, or follicle diameter. Although, there was a tendency for the interaction of treatment by age on follicle size (P = 0.07), with cows having larger follicles than heifers in the no CIDR group but not the CIDR group. In conclusion, use of a CIDR in this resynchronization protocol increased estrus expression, increased AI2 pregnancy for heifers, but did not improve pregnancies in cows, and did not influence overall pregnancy or embryonic loss.