María Alejandra Stornelli

Suppression of estrus in cats with melatonin implants

The objective of this study was to assess the efficacy of a subcutaneous melatonin implant to suppress estrus in queens (felis catus). The hypothesis was that this implant would temporarily and reversibly suppress estrus in queens without producing any clinically detectable side effects. Fourteen adult queens were maintained in cages under artificial illumination (14h light:10h dark) for 45 d and then randomly assigned to one of two treatments. At interestrus, queens received a single subcutaneous melatonin implant (18mg; Melovine [CEVA Sante Animal, Libourne, France]; MEL: n=9), or a single subcutaneous placebo implant without melatonin (0mg; PLA; n=5). At the next estrus, all queens received a second MEL (n=9) or PLA (n=5) implant. Blood samples were taken when queens displayed estrous signs and during interestrus to measure estradiol (E(2)) and progesterone (P(4)), respectively, by radioimmunoassay. There were no significant differences in duration of the interestrus interval in PLA cats, regardless of whether the implants were placed during interestrus or estrus (6.0+/-9.7 d vs. 6.0+/-9.7 d, respectively; least square means [LSM]+/-SEM). However, when MEL implants were placed during interestrus, the duration of interestrus was approximately twice as long as that occurring when MEL implants were placed during estrus (113.3+/-6.1 d vs. 61.1+/-6.8 d, respectively; P<0.01). Serum E(2) and P(4) concentrations were similar in queens with PLA and MEL implants and in queens that received implants in estrus and interestrus. In conclusion, a subcutaneous MEL implant effectively and reversibly suppressed estrus in queens for approximately 2 to 4 mo with no clinically detectable side effects.

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 Bouins 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.

Efficacy of cloprostenol or aglepristone at 21-22 and 35-38 days of gestation for pregnancy termination in queens.

The objective of this study was to evaluate the efficacy of cloprostenol (CLO) or aglepristone (ALI) for pregnancy termination in queens at 21-22 and 35-38 days of gestation. Two experiments (EXP) were carried out to accomplish this aim. Thirty-seven 12- to 14-month-old mixed breed queens were used in a randomized design. At oestrus, queens were housed for mating with a tom, and pregnancy was confirmed by transabdominal ultrasonographic examination (US). On days 21-22 of pregnancy (EXP1) or 35-38 of pregnancy (EXP2), queens were divided into three groups (G). Queens in G1 received ALI (10 mg/kg, sc; EXP1, n = 6; EXP2, n = 6) on two consecutive days. Queens in G2 received CLO (5 μg/kg, sc; EXP1, n = 6; EXP2 = 7) on three consecutive days. Queens in G3 received 1 ml of saline solution (PLA, sc; EXP1, n = 6; EXP2 = 6). After treatment, females were monitored daily by US during for 10 days and weekly until the end of gestation. In EXP1, pregnancy was terminated in (6/6, 100%), (0/6, 0%) and (0/6, 0%), for the ALI, CLO and PLA groups, respectively (p < 0.001). In EXP2, pregnancy was terminated in (6/6, 100%), (1/7, 14%) and (0/6, 0%) for the ALI, CLO and PLA groups, respectively (p < 0.001). In both EXP, after CLO administration, animals vomited and were depressed for 30 min; but no side effects were observed in the animals in the ALI group. In conclusion, the results from this study indicate that three injections of CLO are not effective, but two injections of ALI are effective to induce abortion in queens at 21-22 or 35-38 days of pregnancy.

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.

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.

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 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.

Ultrasonographic and progesterone changes during Days 21 to 63 of pregnancy in queens.

Ultrasonography has been used to diagnose and monitor pregnancy. However, in the queen, most of ultrasonographic and endocrinological studies have been performed using small number of observations during limited periods of pregnancy. The aim of this study was to derive equations to predict the gestation age and parturition time using ultrasonographic embryo fetus measurements and serum progesterone (P4) concentration measurements. Mixed-breed queens (n = 16), aged between 24 and 36 months and weighing between 2 and 4 kg, were daily monitored by ultrasonography since 21 days after the first mating to parturition. Gestational sac (GS) was measured from longitudinal (length [LEN], anterior-posterior [ATP]) and transverse images (width [WID]), GS volume was calculated by the prolate ellipse formula, and GS diameter was calculated by orthogonal measurements. Fetal measurements included crown-rump length (CRL), head diameter (HD), and body diameter (BD). Gestational sac, fetal measurements, and serum P4 concentration were recorded and analyzed by ANOVA. Correlation and linear regression analyses were performed and equations were derived to estimate predicted values and 95% confidence interval for GS parameters and P4 concentrations from 21 to 63 days after the first mating and to estimate predicted values and 95% confidence interval for fetal parameters from Day 35 to 63 of gestation. The average concentrations of serum P4 concentration from Day 22 to 47 of gestation remained between 32.27 ± 4.25 and 16.25 ± 2.45 ng/mL. After that, a gradual decline occurs reaching a concentration of 2.99 ± 1.29 ng/mL 1 day before parturition. A positive and significant correlation between the ultrasonographic measurements (LEN, ATP, WID, GS volume and diameter, uterine wall thickness, CRL, HD, and BD) with number of days after the first mating was observed (P < 0.001). We observed a positive and significant correlation between GS measurements (LEN, ATP, and WID) and between fetal measurements (CRL, HD, and BD) and a negative and significant correlation between serum P4 concentration with GS (LEN, ATP, and WID), uterine wall thickness, and fetal (CRL, HD, and BD) measurements. In addition, there was a positive and significant correlation between serum P4 concentrations with days after the first mating to parturition. In conclusion, the equations derived from this study will be useful for pregnancy monitoring and for estimating pregnancy age in queens from Day 21 until parturition for animals with similar weight and age.

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.

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.