Effect of natural photoperiod on epididymal sperm quality and testosterone serum concentration in domestic cat (Felis silvestris catus).

The aim of this study was to assess epididymal sperm characteristics and serum testosterone concentration in cats under natural photoperiod. The hypothesis was that natural photoperiod induces seasonal changes in spermatozoal quality and serum testosterone concentration. Mixed breed tomcats (n = 43) that underwent bilateral orchiectomy at a municipal public pet shelter were used in the study. Epididymides were divided into two groups according to time of castration. In Group I, toms were castrated during increasing light (IL; [winter and spring; n = 24]), and group II, during decreasing light (DL; [summer and fall; n = 19]). Only mature toms castrated in the two lasts weeks of each season were included in this study. Sperm samples were obtained by cutting the cauda epididymis in Tris solution and tested for motility (MOT,% motile), velocity (VEL, 0-5), total sperm cells (TS, 10(6) ), acrosome integrity (ACR,% intact; FITC-PSA), plasma membrane integrity (MI,%intact; CFDA-PI) and sperm morphology (SM,% normal). Before orchiectomy, blood samples were taken to measure serum concentrations of testosterone (T2) by a solid-phase RIA. Data were analysed with the mixed procedure of SAS. Toms castrated during IL had higher sperm plasma membrane integrity and better sperm morphology compared to toms castrated during DL (69.0 ± 2.7 vs 60.6 ± 2.1, p < 0.01; 45.9 ± 2.5 vs 35.9 ± 3.4; p < 0.02; respectively) and tended to have higher sperm motility and total number of sperm cells compared to toms castrated during DL (56.3 ± 2.8 vs 47.3 ± 3.7, p < 0.06; 13.8 ± 1.4 vs 10.0 ± 1.8, p < 0.09). However, velocity, acrosome integrity and serum testosterone concentrations were similar between both groups (3.5 ± 0.1 vs 3.4 ± 0.1, p > 0.6; 45.8 ± 3.3 vs 44.0 ± 4.0, p > 0.72; 0.76 ± 0.15 vs 0.59 ± 0.19, p > 0.51; respectively). In conclusion, natural photoperiod induces seasonal changes in sperm quality with a moderate variation in serum testosterone concentrations.

Effect of refractoriness to long photoperiod on sperm production and quality in tomcats.

The aim of this study was to assess whether refractoriness to long photoperiod (LP) could be reversed by subjecting tomcats to a period of short days. Our hypothesis was that photoperiod changes can avoid refractoriness and restore sperm quality and production to that before refractoriness. Tomcats (n = 6) were housed in a conditioned room with LP (12L: 12D) for 45 days of acclimation and then maintained under LP for 18 month. Then, tomcats were changed to a period of decreasing light at a rate of 8 min/day for 1 month. Tomcats stayed for 1 month with short photoperiod (SP; 8L: 16D) and then were switched back to a period of increasing light at a rate of 8 min/day for 1 month. The experiment was completed after tomcats remained in LP for 2 months. Toms were anaesthetized and semen samples were collected by electroejaculation every 2 weeks. Sperm parameters were evaluated in all ejaculates, and data were analysed by anova. Motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher during LP compared with a refractory LP (p < 0.01). Likewise, velocity, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher in a LP compared with a SP (p < 0.05). On the other hand, motility, volume, concentration and total sperm count were similar between LP and SP (p > 0.20).Whereas motility, velocity, viability, acrosome integrity and plasma membrane integrity were similar in a refractory LP compared with SP (p > 0.05), volume, sperm concentration, total sperm count and sperm morphology were lower in a refractory LP compared with SP (p < 0.05). In conclusion, refractoriness and reduced sperm production and quality induced by a prolonged LP of 18 month can be restored after placing tomcats to a SP.

Effect of storage media and storage time on histological and ultrastructural changes in cat epididymal cells.

The aim of this study was to assess the histological and ultrastructural changes in cat epididymides (n = 22) stored at 4 °C in two different media [saline solution (SAL) or tris-egg yolk (TEY)]. Our hypothesis was that epididymides stored in TEY would have delayed epithelial cell autolysis. Four epididymides were fixed and processed immediately, and the remaining 18 epididymides were stored at 4 °C in SAL or TEY for 24, 48 or 72 h. In histological sections, the nuclear features and stereocilia morphology were scored from 0 to 3. Ultrastructurally, nuclear chromatin and stereocilia morphology were scored from 0 to 3. In addition, using transmission electron microscopy nuclear number, nuclear area, mitochondrial number and mitochondrial area were recorded. In the histological study, parameters changed with time and media (p < 0.01). A significant effect of time was observed (p < 0.01), and the morphological changes were greatest when the storage time increased. Morphological changes were higher in SAL compared with TEY (p < 0.01). In the ultrastructural study, nuclear chromatin and stereocilia morphology decreased with time and media as in the histological study (p < 0.01). In addition, nuclear number and nuclear area changed with time (p < 0.004; p < 0.001) but not with media. Conversely, mitochondrial number and mitochondrial area did not change with media or time (p > 0.05). In conclusion, these results show that TEY preserved epididymal epithelial cells better than SAL; this finding could help improve sperm quality of stored epididymides.

Progesterone and ultrasonographic changes during aglepristone or cloprosternol treatment in queens at 21 to 22 or 35 to 38 days of pregnancy.

Progesterone (P4) is a requirement for pregnancy development. Previous reports observed a maximal value of serum P4 concentration on 21 days after the first mating after which it slowly declines throughout the rest of pregnancy. Ultrasound examination should be performed to ensure that pregnancy interruption is complete. Limited information is available on the ultrasonic appearance of conceptuses during pregnancy termination in cats The objective was to study serum P4 concentration and ultrasonographic changes during aglepristone (ALI) or cloprostenol (CLO) treatment and to evaluate the fertility after treatment. Two experiments (EXP) were carried out to accomplish this aim. Sixty queens, 12- to 36-month-old, were used. On Days 21 to 22 of pregnancy (EXP I) or 35 to 38 of pregnancy (EXP II), queens were divided into three groups (G). Queens in G1 received ALI (10 mg/kg, sc; EXP I, n = 10; EXP II, n = 10) for 2 consecutive days. Queens in G2 received CLO (5 µg/kg, sc; EXP I, n = 10; EXP II = 10) for 3 consecutive days. Queens in G3 received 1 mL of saline solution (PLA, sc; EXP I, n = 10; EXP II = 10). Blood samples were taken before treatment (Day 0) and every day during 10 days after the treatment to measure serum P4 concentrations. Likewise, after treatment, queens were monitored daily by ultrasonography for 10 days and weekly until the end of gestation to obtain gestational sacs measurements (GS), fetal measurements, and fetal biophysical profile. Data were analyzed by ANOVA. Serum P4 concentrations were significantly different on Day 6 (EXP I) and on Day 1 (EXP II) in ALI and CLO groups compared with PLA group (P < 0.05 and P < 0.01; respectively). The ultrasonographic monitoring during treatment allowed assessing changes in the GS and fetal measurements, embryo-fetal viability, and risk of pregnancy loss. In conclusion, the results from this study reported changes in serum P4 concentration and in ultrasonography measurements during pregnancy interruption with ALI or CLO treatment. Also it was observed that ALI and CLO are safe drugs and can preserve posttreatment queen fertility. Therefore, the results obtained in our work will be applied in feline reproduction practice.

Effect of melatonin implants on spermatogenesis in the domestic cat (Felis silvestris catus).

The aim of this study was to assess the efficacy of subcutaneous melatonin implants to temporarily and reversibly suppress spermatogenesis in male cats. Tomcats (n = 8) were housed in a conditioned room with alternating long and short 2-month photoperiod cycles to maintain sperm production and quality. Animals were randomly assigned to one of the two treatments. Four animals received a subcutaneous melatonin implant (MEL, 18 mg; Syntex, Argentina), whereas the other four received a subcutaneous placebo implant (PLA, 0 mg; Syntex). Semen samples were collected by electroejaculation every 14 days for 252 days. Sperm parameters were evaluated in all ejaculates, and data were analyzed by ANOVA. Melatonin-implanted cats significantly decreased their sperm quality in all the parameters studied compared with the control group (MEL vs. PLA; least squares means ± SEM; motility, 71.3 ± 3.4 vs. 82.1 ± 3.6; velocity, 3.4 ± 0.1 vs. 4.6 ± 0.1; total sperm count, 2.6 ± 2.2 vs. 19.4 ± 3.3; acrosome integrity, 48.7 ± 5.6 vs. 62.8 ± 5.6; plasma membrane integrity, 52.2 ± 4.7 vs. 72.9 ± 5.5; normal sperm morphology, 45.8 ± 3.3 vs. 63.7 ± 3.4; P < 0.05). Conversely, volume and serum testosterone concentrations were similar in both groups (volume, 0.15 ± 0.02; serum testosterone concentrations, 1.1 ± 0.1; CV 18.9%; P > 0.05). At 91 ± 7 days after implant insertion, sperm motility decreased 38.5%, velocity 26.5%, total sperm count 82%, acrosome integrity 22%, plasma membrane integrity 30%, and normal sperm morphology decreased 32% of preimplant values. This effect was present until 120 ± 15 days after implant insertion. After that, seminal parameters started to increase and reached preimplant values at about 140 ± 7 days after implant insertion. Nevertheless, treated animals conserved the capacity to produce semen during the treatment period. In conclusion, a single subcutaneous melatonin implant effectively and reversibly reduced sperm production and quality in male domestic cats for approximately 120 ± 15 days without clinically detectable adverse effects.

Pharmacokinetics of eCG and induction of fertile estrus in bitches using eCG followed by hCG.

The aim was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment 1, three ovariohysterectomized bitches received 10 000 IU of eCG iv, and 15 days later 10 000 IU of eCG im. Blood samples were taken up to 144 h after each injection to measure eCG concentrations. In Experiment 2, 25 healthy, intact late anestrous bitches were assigned to one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Sexual behavior (SB), clinical signs of estrus (CSE) and vaginal cytology (VC) samples were obtained and scored before eCG administration and every other day until onset of estrus, or for 14 days. In Experiment 3, intact late anestrous bitches were assigned to a treatment group (TRT; n = 16) and received eCG (50 IU/kg im) followed by hCG (500 IU im) 7 days later; or to a placebo group (PLA; n = 8) where they received 1 mL saline solution im. All bitches that were induced in estrus were mated or AI with fresh semen. In Experiment 1, maximum observed concentration (C(max)) eCG were similar between im and iv routes (6.1 ± 0.9 vs. 8.6 ± 0.5 IU/mL, P > 0.08), whereas time for maximum observed concentration (T(max.)) was longer for im compared to iv routes (17.5 ± 0.5 vs. 11.6 ± 0.3 h, P < 0.01). The area under the curve (AUC) was similar for im and iv routes (P > 0.48), and eCG was detectable in serum for at least 144 h for both routes. In Experiment 2, 3 days or 3 to 5 days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, the mean interval from treatment to estrus was 4.0 ± 0.4 days. In Experiment 3, the mean interval from treatment to estrus was shorter in the TRT group compared to the PLA group (4.1 ± 3.3 vs. 68.5 ± 4.4 days, P < 0.01). The previous interestrus interval was similar for TRT and PLA groups (199.6 ± 7.2 vs. 197.5 ± 10.2 days), but the new interestrus interval was shorter for the TRT compared to the PLA group (164.0 ± 7.2 vs. 212.2 ± 10.2 days; treatment by interval interaction, P < 0.007). Serum P(4) concentrations increased on the first day of cytologic diestrus after treatment in bitches in TRT (0.7 ± 0.3 vs. 22.8 ± 4.2 ng/mL; P < 0.01); but did not change in PLA (P > 0.84). Ninety-four percent of animals were bred (15/16; AI, n = 7; natural mating, n = 8), and 80% (12/15) became pregnant. None of the bitches had any side effects from the eCG and hCG therapy. We concluded that 50 IU/kg of eCG combined 7 days later with 500 IU of hCG was effective to induce normal and fertile estrus in bitches at 164 days post estrus, with an 80% pregnancy rate, with no side effects, and with a reduction of 48 days of the interestrus interval.