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 degrees 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 degrees 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. (C) 2022 The Authors. Published by Elsevier Inc.

Introduction: This study evaluated whether post in vitro capacitation changes in sperm could be used to estimate field fertility differences between bulls.Methods: Frozen-thawed semen from five bulls (two to four ejaculates per bull) previously identified as high (48.1% and 47.7%), intermediary (45.5%) or low (40.7% and 43.1%) fertility, based on pregnancy per AI (P/AI), were evaluated for total and progressive motility, sperm plasma membrane integrity (viability), acrosome integrity (viable sperm with an intact or disrupted acrosome), reactive oxygen species (ROS; viable sperm ROS+ or ROS-), mitochondrial membrane energy potential, zinc signatures (signatures 1-to-4) and CD9 protein populations at pre-wash and post-wash (only total and progressive motility), h0 (diluted with non-capacitation media), and at h0, h0 CM, h3, h6, and h24 after dilution with capacitation media (CM) and incubation at 37ºC. Data were analyzed using the GLIMMIX procedure as repeated measures in SAS with bull, time and the interaction as fixed effects.Results: Bull by time interaction was significant (P≤0.03) for total motility, viability, viable sperm with disrupted acrosome, and zinc signature 3. There tended (P=0.06) to be a bull by time interaction for zinc signatures 1+2 combined. Time was significant (P≤0.003) in all analyses, except viable ROS- (P=0.12). There was a significant effect of bull (P≤ 0.03) for viability, viable sperm with disrupted acrosome, zinc signatures 1, 2 and 1+2, viable CD9- and dead CD9+. High and intermediary fertility bulls had greater (P≤0.04) percentages of viable sperm, zinc signature 2 and zinc signature 1+2 compared to low fertility bulls. High and intermediary fertility bulls had decreased (P≤0.05) percentage of dead CD9+ compared to low fertility bulls. Viable CD9+ differed (P=0.02) and viable sperm with an intact acrosome and viable CD9+ tended to differ (P=0.06) amongst bulls; however, association with field fertility was not observed. There was a positive correlation between P/AI and zinc signature 2 (P=0.04), and there tended to be a positive correlation between P/AI and viability (P=0.10), and zinc signature 1+2 (P=0.10).Discussion: In summary, incubation of sperm in CM and flow cytometry analyses for viability, zinc signatures 2 and 1+2, and dead CD9+ seems promising to estimate in vivo fertility differences amongst bulls.

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.

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).

Transrectal ultrasonography is known as the gold standard for pregnancy detection, but requires costly equipment and technical skills; therefore, access to an inexpensive and more user-friendly method with similar accuracy could benefit cattle producers. Detection of pregnancy-associated glycoproteins can accurately determine pregnancy in ruminants; however, usually requires specialized equipment for the assay. Thus, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. In study 1, blood samples from previously identified pregnant Bos taurus females from six different herds (nulliparous n = 1,205 and multiparous n = 1,539; samples collected between d 27 to 285 of gestation over a three-year period) were utilized. In study 2, postpartum females (primiparous n = 48 and multiparous n = 66) from one herd were utilized: (n = 1,066; samples collected weekly for up to 12 weeks postpartum). In study 1, level of agreement between different methods of pregnancy detection was determined by Pearson’s correlation and Kappa scores. In study 2, data were analyzed as a repeated measure using the MIXED procedure of SAS with main effects of parity, days postpartum (dpp), and parity by days postpartum, then data were analyzed further using the REG procedure of SAS. In study 1, transrectal ultrasonography and lateral flow were positively correlated (r = 0.77; P <0.01), with 92.4% agreement. In study 2, the abundance of absorbance of PAGs rapidly decreased from 0 to 50 days postpartum, then continued to gradually decrease (P <0.01; r = 0.90). Prior to 42 days postpartum, PAG concentrations were sufficiently elevated resulting in false positive readings in all assays. In conclusion, there is very good agreement between transrectal ultrasonography and PAG assays, but likelihood of false positive results are highif assays are performed fewer than 42 days postpartum.