Cryopreservation of canine epididymal sperm using ACP-106c and TRIS.

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P<0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12h of cooling for both extenders (P<0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.

Proteomic characterization of canine seminal plasma.

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at -80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.

Semen quality, testicular B-mode and Doppler ultrasound, and serum testosterone concentrations in dogs with established infertility.

Retrospective examination of breeding records enabled the identification of 10 dogs of normal fertility and 10 dogs with established infertility of at least 12 months of duration. Comparisons of testicular palpation, semen evaluation, testicular ultrasound examination, Doppler ultrasound measurement of testicular artery blood flow, and measurement of serum testosterone concentration were made between the two groups over weekly examinations performed on three occasions. There were no differences in testicular volume (cm(3)) between the two groups (fertile right testis = 10.77 ± 1.66; fertile left testis = 12.17 ± 2.22); (infertile right testis = 10.25 ± 3.33; infertile left testis = 11.37 ± 3.30), although the infertile dogs all had subjectively softer testes compared with the fertile dogs. Infertile dogs were either azoospermic or when they ejaculated, they had lower sperm concentration, sperm motility, and percentage of morphologically normal spermatozoa than fertile dogs. Furthermore, infertile dogs had reduced sperm membrane integrity measured via the hypoosmotic swelling test. Infertile dogs had significantly lower basal serum testosterone concentrations (1.40 ± 0.62 ng/mL) than fertile dogs (1.81 ± 0.87 ng/mL; P < 0.05). There were subjective differences in testicular echogenicity in some of the infertile dogs, and important differences in testicular artery blood flow with lower peak systolic and end-diastolic velocities measured in the distal supratesticular artery, marginal testicular artery, and intratesticular artery of infertile dogs (P < 0.05). Notably, resistance index and pulsatility index did not differ between infertile and fertile dogs. These findings report important differences between infertile and fertile dogs which may be detected within an expanded breeding soundness examination.

Favoring the birth of female puppies after artificial insemination using chilled semen diluted with powdered coconut water (ACP-106c).

The objective was to determine the effect of powdered coconut water extender (ACP-106c) on the proportion of female puppies born. Twenty French Bulldog bitches were subjected to natural mating (NM) and, during the subsequent two estrus periods, were bred by intravaginal artificial insemination (AI), using chilled semen (from the same males) diluted in Tris-egg yolk (AI-Tris) or ACP-106c (AI-ACP-106c). Fresh semen was cooled to 5 °C and maintained at that temperature for 6 h, rewarmed (37 °C for 30 s), and used for AI. Pregnancy and whelping rates following NM were both 100% and were both 90.0% following AI with either extender. Litter size (mean ± SD) was 5.4 ±1.1, 4.7 ± 2.0, and 5.1 ± 2.0 (P > 0.05) for NM, AI-Tris, and AI-ACP-106c, respectively. Furthermore, for these groups, the number of female vs. male puppies born were 2.6 ± 0.6 vs. 2.8 ± 1.0, 2.2 ± 1.0 vs. 2.5 ± 1.1, and 3.4 ± 1.6 vs. 1.8 ± 1.2 (P < 0.05 for AI-ACP-106c only). In conclusion, our hypothesis was supported; AI of semen in ACP-106c extender resulted in a significantly higher proportion of female puppies. Furthermore, this extender yielded acceptable litter size and rates of pregnancy and whelping.