Heat stress and sperm production in the domestic cat.

This study aimed to assess 1) the effect of high environmental temperatures on sperm production and 2) the effectiveness of a temperature-humidity index (THI) to predict the degree of thermal stress in a cat model. Semen collection was performed by electroejaculation for 18 mo in 20 tomcats maintained under controlled photoperiod. Still, temperature and humidity were not experimentally manipulated to describe the effect of natural climate conditions on seminal samples. Ejaculates (n = 512) were then grouped according to temperature records of the sampling day and compared by temperature and THI index. Significant lower sperm parameters and increase sperm tail abnormalities were observed during warm environments (temperature and THI). Concentration and total sperm count were the most affected parameters. Environmental temperatures of 28.5 °C with 54% relative humidity (THI = 77.07) and 27.9 °C with 66% humidity (THI = 77.84) were upper thresholds of moderate thermal stress. Moreover, days with relative humidity near 90% led to severe thermal stress with temperatures as low as 26.6 °C (THI = 78.88). The current study demonstrates the detrimental effect of high environmental temperatures on sperm quality in the domestic cat. This effect is observed at lower temperatures when high relative humidity is present. In this sense, the THI was a reliable predictor of the magnitude of thermal stress experienced by cats. Thus, cats from reproductive programs should be maintained under controlled photoperiod cycles with temperatures around 20 °C and humidity around 70% to avoid semen detrimental effects.

Effect of cat seminal plasma and purified llama ovulation-inducing factor (ß-NGF) on ovarian function in queens.

The present study aimed to determine the effect of cat seminal plasma and purified llama ovulation-inducing factor (ß-NGF) on ovarian activity in queens. Queens (n = 6) were used for all the treatments in a crossover design with an interval time between treatments of three interestrus intervals. Forty-eight hours after the detection of an estrus vaginal cytology, queens were given cat seminal plasma (subcutaneous or intramuscular), purified llama ovulation-inducing factor (15 or 35 µg), hCG (75 UI), saline, or were mated with a male. A total of 192 estrous cycles were observed. Estrus length and serum estradiol concentration were 6 ± 1 days (range 2-10 d) and 38 pg/mL (range 10-75 pg/mL), respectively. Queens mated and given hCG showed higher serum progesterone concentration and longer interestrus interval (47 ± 5 d) than that of controls (10 ± 3 d). Sixty-seven percent of queens (4/6) treated with subcutaneous cat seminal plasma, and 17% of those treated with purified llama ß-NGF showed high serum progesterone concentrations along with prolonged interestrus. However, intramuscular administration of cat seminal plasma produced interestrus intervals similar to controls (15 ± 5 d) and basal serum progesterone concentration (<0.50 ng/mL). This study demonstrates that the subcutaneous administration of cat seminal plasma induced ovulation in queens. Therefore, molecules present in cat seminal plasma, contribute to the induction of ovulation in queens. Identifying those molecules will improve the knowledge of queen's reproductive physiology. Also, it could offer a physiologic alternative to induce ovulation in queens when reproductive biotechnologies are used.

Uterine endometrial cytology, biopsy, bacteriology, and serum C-reactive protein in clinically healthy diestrus bitches.

The aim of this work was to assess the agreement between endometrial cytology and uterine biopsy for the diagnosis of endometritis (END), the bacterial populations isolated from the vagina and uterus of bitches having END, and the measurement of C- reactive protein as a diagnostic tool for diagnosis of END in clinically healthy bitches. Fifty privately-owned intact, clinically healthy bitches, in diestrus, without a history of progestin administration, aged between 8 months and 6 years old and weighing between 5 and 28 kg were used in this study. Bitches were included in a program for breeding control at a municipal pet public shelter. Before ovariohysterectomy (OVX) samples for vaginal cytology and bacteriology, and blood samples were taken. After OVX endometrial cytology, bacteriological samples and biopsy were collected. Histologic examination was performed to confirm the uterine condition. Blood samples were centrifuged and stored at -20 °C until progesterone (P4) and C-reactive protein was measured. Samples for bacteriological culture were collected, and swabs were placed into Stuart's transport medium and transported to the laboratory. On histopathologic examination, the most common observation was END (27/50), followed by normal endometrium (NE; 18/50), cystic endometrial hyperplasia (CEH; 2/50), atrophy (2/50) and fibrosis with degeneration of the endometrial glands (1/50). Low degree of agreement was observed between results obtained by endometrial cytology samples and results obtained by biopsy in endometrial diagnosis (Kappa Coefficient: -0.19). In vaginal samples, ß-hemolytic Streptococcus, Staphylococcus spp., Escherichia coli, Proteus spp., Corynebacterium spp., and Klebsiella pneumoniae were the bacteria most often found. In uterine samples, only four samples from END showed bacterial grow. C-reactive protein frequency was higher in END (6/23, 23%) vs NE (0/16, 0%; Van der Waerden P-value = 0,0302). Our results support the hypothesis that END is a frequent finding in uterine biopsy and could be associated with subfertility and infertility in the bitch. A low degree of agreement was observed between the diagnostic results from the uterine biopsy and endometrial cytology. Bacteriology would not be recommended as a diagnostic tool because no bacteria highly associated with uterine diseases were isolated from bitches with END. Finally, the usefulness of C-reactive protein concentration as a marker for END in bitches could not be conclusively demonstrated.

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.

Prolactin, Androstenedione and IGF1 Serum Concentrations During Induced Follicular Growth by eCG Administration in the Bitch.

The oestrus cycle in the domestic bitch, a monoestrous species, differs considerably from that of other veterinary domestic animals species. In the bitch the combined use of eCG and hCG is effective to induce oestrus predictably and safely (Stornelli et al., Theriogenology, 78, 2012 and 1056). Although several studies were done to describe the hormonal changes during the canine oestrus cycle, to our knowledge none was done to describe the hormonal changes during induced follicular growth after the administration of eCG. The aim of this work was to study prolactin (PRL), insulin-like growth factor (IGF1) and androstenedione (ANDR) serum concentrations during follicular growth induced by a single dose of eCG administered to late anoestrous bitches. PRL and ANDR concentrations were lower before than after eCG TRT (before eCG vs pro-oestrus, oestrus and dioestrus; 4.3 ± 1.8 ng/ml vs 6.5 ± 1.6 ng/ml, p < 0.05; 0.08 ± 0.2 ng/ml vs 0.42 ± 0.16 ng/ml, p < 0.05). Conversely, IGF1 concentrations were similar before and after eCG TRT (286.0 ng/ml ±32.2, p > 0.53). Additionally, PRL concentrations were similar before oestrus compared to during oestrus and dioestrus (6.9 ± 1.7 ng/ml, p > 0.19). Furthermore, IGF1 concentrations were higher before and during oestrus compared to first day of dioestrus (286.1 ± 29.8vs 200.4 ± 29.2 ng/ml, p < 0.01). On the contrary, ANDR concentrations were lower before and during oestrus compared to first day of diestrum (0.35 ± 0.17 ng/ml and 0.38 ± 0.15 vs 0.68 ± 0.17 ng/ml, p < 0.05). These results show that treatment with a single injection of 50 IU/kg of eCG in late anoestrous bitches successfully induced changes in follicular growth which were paralleled with changes in PRL, IGF1 and ANDR serum concentration similar to those occurring during a normally occurring oestrous cycle. In addition, our results suggest that IGF1 in the bitch could play an important role in ovarian folliculogenesis.

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.

Seasonal changes in testicular cell morphology in domestic male cats (Felis catus).

The aim of this study was to asses the variation in the morphology of the seminal epithelium in relation to natural photoperiod in male cats. Tom cats (n = 240) were castrated every other week throughout the year. Each testis was fixed in Bouin's solution and cut into sections. The percentage of tubules with round spermatids (RS), elongated spermatids (ES), tailed spermatids (TS), mature spermatids (MS) and the number of Sertoli cells (SC) and Leydig cells (LC) were recorded in each sample. Testicles from males during short days (SHD) had a higher percentage of tubules with RS and ES compared to testicles from males during long days (LHD, 31.3 +/- 0.6 vs 2.1 +/- 0.6%, p < 0.001; 30.9 +/- 0.7 vs 11.0 +/- 0.7%, p < 0.001). Conversely, testicles from males during SHD had a lower percentage of tubules with TS and MS compared to testicles from males during LHD (24.5 +/- 0.8 vs 29.7 +/- 0.8%, p < 0.01; 13.1 +/- 1.2 vs 57.0 +/- 1.2%, p < 0.01). Furthermore, testicles from males during SHD had a higher number of SC and lower number of LC compared to testicles from males during LHD (11.4 +/- 0.1 vs 8.0 +/- 0.1%, p < 0.01; 19.2 +/- 1.0 vs 38.0 +/- 1.0%, p < 0.01). In conclusion, there are seasonal changes in testis cell morphology in the tom which may be related to seasonal sperm production.