ProAKAP4 Semen Concentrations as a Valuable Marker Protein of Post-Thawed Semen Quality and Bull Fertility: A Retrospective Study.

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls' semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d (p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility.

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos.

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ±â€¯9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ±â€¯0.19 × 103 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.

The benefits of liposomes for chilling canine sperm for 4 days at 4°C.

This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4°C) canine sperm over a period of 4 days. In the first experiment, 20 ejaculates collected from 5 Beagle dogs were chilled in an extender containing 6% low density lipoproteins (LDL) (Control), or one of 7 extenders containing different concentrations (2, 4, 6, 8, 10, 15, 20%) of liposomes (LIPO). These ejaculates were chilled over 4 days and motility was assessed daily using a Hamilton Thorne analyzer (HTM-IVOS, 14.0). The 2% LIPO obtained the best results (p=0.038) after four days (72.55% motile spermatozoa and 31.4% progressive spermatozoa). In experiment 2, 10 ejaculates were collected from same 5 dogs and chilled in 6% LDL or 2% LIPO-based extenders. Sperm integrity characteristics were assessed prior to refrigeration and every 48h for four days (D0, D2, and D4). Acrosome integrity was assessed using the FITC-PSA test (Fluorescein IsoThiocyanate-Pisum Sativum Agglutinin), plasma membrane (PM) integrity using both the hypo-osmotic swelling test (HOSt) and SYBR14/Propidium Iodide test (SYBR14/PI), and DNA integrity using the Acridine-Orange test (AO). The 2% LIPO extender provided equivalent preservation of sperm integrity parameters to the reference extender (6% LDL). In experiment 3, a Langmuir-Blodgett trough was used to evaluate the mechanistic interactions between LDL, LIPO, prostatic fluid, and the canine spermatozoal membrane during chilling. Results indicate that LDL and LIPO interact differently with the biomimetic membrane. The most likely conclusion of these findings is that LDL and liposomes employ different protective mechanisms during the chilling (4°C) of canine spermatozoa.