Determination of Appropriate Cryopreservation Protocols for Epididymal Cat Spermatozoa

Effects of Equex and glycerol additions and sample dilution step on frozen–thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one‐ (groups III and IV) or two‐ (groups I, II, V and VI) step dilution. For one‐step dilution, the pellets were resuspended in plain egg yolk‐Tris medium (EYT) + 5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4^°C, 1 h). For two‐step dilution, the pellets were resuspended in EYT (I and V) and in EYT + 3% glycerol (II and VI), cooled and further diluted with EYT + 10% glycerol with (I)/without (V) 1% Equex and with EYT + 7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze–thawing, no differences (p > 0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p = 0.005 to p = 0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p > 0.05) and was higher than the rest (p < 0.001 to p = 0.02). At 2 h after thawing, the motility, viability and HOST were comparable among the groups (p > 0.05) except the lower percentages of viability in III (p = 0.008 to p = 0.3) and of HOST in IV (p = 0.005 to p = 0.2). Two‐step dilutions with Equex (I, II) were more beneficial for the FITC at 2 h than without Equex (V) (p = 0.005 and p = 0.02) and than one‐step dilutions (III, IV) (p < 0.001 to p = 0.02). In conclusion, epididymal cat sperm quality after freeze–thawing could be improved when Equex was added and two‐step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.     

Intra-uterine insemination with low numbers of frozen–thawed boar spermatozoa in spontaneous and induced ovulating sows under field conditions

The present study was conducted to evaluate non-return rate (NR), farrowing rate (FR), and number of total pigs born/litter (TB) of weaned sows after intra-uterine insemination (IUI) using low numbers of frozen–thawed (FT) spermatozoa. Semen from 6 boars was cryopreserved individually in a 0.5-ml straw, at a concentration of 1 × 10⁹ spermatozoa/ml. A total of 40 multiparous sows with weaning-to-estrus interval of 3 to 7 days were included. The sows were detected for standing estrus twice daily and randomly assigned to two groups: I) spontaneous ovulation (n = 20) and II) induced ovulation (n = 20) which the sows were given 750 IU human chorionic gonadotrophin (hCG) i.m. immediately at estrus detection. Ovulation was determined every 12 h using transrectal ultrasonography. FT semen containing 1 × 10⁹ motile spermatozoa/dose was used to IUI. In group I, the sows were inseminated at 24 h after the detection of estrus and repeated every 12 h until ovulation. In group II, the sows were inseminated at 36, 42 and/or 48 h after hCG treatment. The results showed that the interval from standing estrus to ovulation (EOI) differed significantly between group I (40.2 h) and group II (35.6 h; P = 0.01). Variation of EOI among sows within each group seemed to be lower in group II (4.5 h SD) than in group I (5.5 h SD; P = 0.5). The number of IUI per sow was 2.9 ± 0.6 times in group I and was 2.4 ± 0.5 times in group II. There were no significant differences (P > 0.05) in the NR (80 vs 85%), FR (60 vs 65%) and the TB (8.0 ± 2.8 vs 9.4 ± 3.7 piglets/litter) between the groups. These results indicated that multiple doses of IUI with a low number of FT boar spermatozoa provided a fairly good NR, and reasonable FR and TB both in spontaneous and induced ovulating sows. The number of inseminations required for attaining acceptable fertility tended to be lower in the weaned sows with induced ovulation.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

Sperm‐TALP: An Alternative Extender for Retrieving and Diluting Epididymal Sperm in the Domestic Cat

Effects of sperm‐TALP (TALP) on the quality of fresh‐extended and frozen‐thawed epididymal cat sperm were evaluated. The epididymides suspended in Tris–glucose–citrate solution (Tris), a conventional medium, and TALP were cut into small pieces to recover epididymal sperm. In experiment 1, the sperm pellets remained after centrifugation were re‐suspended (1 : 2, v/v) in Tris and TALP. The sperm quality in all four groups, that is, sperm retrieved with Tris (I and II) or TALP (III and IV) and diluted with Tris (I and III) or TALP (II and IV) was assessed. The sperm motility at the 0‐h incubation in TALP–TALP was superior to that of the rest (p < 0.001 to p = 0.04). At the 2‐h incubation, the motility in Tris/TALP–TALP was greater than that in Tris/TALP–Tris (p ≤ 0.001). In experiment 2, after centrifugation, the sperm pellets were added with freezing extenders and frozen. The thawed sperm previously retrieved from the epididymides with Tris and TALP were allotted so as not to further diluted (Tris/TALP–O) and to further diluted (1 : 1, v/v) with Tris (Tris/TALP–Tris) and TALP (Tris/TALP–TALP) and were evaluated the quality. At both incubation times, the motility of frozen‐thawed sperm recovered with TALP (TALP–O/Tris/TALP) was comparable with or significantly higher than that in the Tris groups (Tris–O/Tris/TALP; p = 0.003 to p > 0.05). The motility and viability of thawed sperm in Tris–Tris were significantly decreased during the 2‐h incubation (p = 0.007 for the motility and p = 0.01 for the viability). In both experiments, neither type of diluent (Tris vs TALP) nor incubation time (0 vs 2 h) significantly affected the sperm membrane integrity under hypo‐osmotic condition (p > 0.05). According to beneficial effects on the quality of fresh‐extended and frozen‐thawed sperm demonstrated, sperm‐TALP could be used as an alternative medium for recovering sperm from the epididymides and for diluting epididymal sperm in the domestic cat.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Effect of the administration of GnRH or hCG on time of ovulation and the onset of estrus-to-ovulation interval in sows in Thailand

The aim of the present study was to evaluate the control of ovulation by the administration of human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone (GnRH) at the onset of estrus. Thirty-three multiparous sows housed under tropical conditions and showing standing estrus within 5 days after weaning were included. The sows were allocated to three groups, spontaneous ovulation (control group, n = 10), induced ovulation using 750 IU hCG (hCG group, n = 10), and induced ovulation using 50 μg GnRH (GnRH group, n = 13). The hormones were given at the onset of estrus and the occurrence of ovulation was monitored every 6 h by transrectal ultrasonography. Data for weaning-to-estrus interval, onset of estrus-to-ovulation interval (EOI), and the length of estrus were recorded. All sows in the control and hCG groups ovulated, while 3 out of 13 sows treated with GnRH developed cystic ovaries (did not ovulate). Of those sows ovulating, the EOI of the hCG (40.2 ± 1.7 h) and GnRH (37.5 ± 3.3 h) groups were shorter than that of the control group (63.6 ± 9.6 h; P < 0.05). In conclusion, the administration of either hCG or GnRH at the onset of estrus can control time of ovulation but, at the dose employed, sows receiving GnRH may develop ovarian cysts.     

Prevalence of autoantibodies that bind to kidney tissues in cats and association risk with antibodies to feline viral rhinotracheitis,calicivirus, and panleukopenia

BackgroundThe feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine, prepared from viruses grown in the Crandell-Rees feline kidney cell line, can induce antibodies to cross-react with feline kidney tissues.ObjectivesThis study surveyed the prevalence of autoantibodies to feline kidney tissues and their association with the frequency of FVRCP vaccination.MethodsSerum samples and kidneys were collected from 156 live and 26 cadaveric cats. Antibodies that bind to kidney tissues and antibodies to the FVRCP antigen were determined by enzyme-linked immunosorbent assay (ELISA), and kidney-bound antibody patterns were investigated by examining immunofluorescence. Proteins recognized by antibodies were identified by Western blot analysis.ResultsThe prevalences of autoantibodies that bind to kidney tissues in cats were 41% and 13% by ELISA and immunofluorescence, respectively. Kidney-bound antibodies were observed at interstitial cells, apical border, and cytoplasm of proximal and distal tubules; the antibodies were bound to proteins with molecular weights of 40, 47, 38, and 20 kDa. There was no direct link between vaccination and anti-kidney antibodies, but positive antibodies to kidney tissues were significantly associated with the anti-FVRCP antibody. The odds ratio or association in finding the autoantibody in cats with the antibody to FVRCP was 2.8 times higher than that in cats without the antibody to FVRCP.ConclusionsThese preliminary results demonstrate an association between anti-FVRCP and anti-cat kidney tissues. However, an increase in the risk of inducing kidney-bound antibodies by repeat vaccinations could not be shown directly. It will be interesting to expand the sample size and follow-up on whether these autoantibodies can lead to kidney function impairment.