Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

The significance of small vs large intestinal digestion of cereal grain and oilseed protein in the equine

Mature ponies fitted with permanent ileal cannulas were used in two 3×3 Latin square experiments to quantify prececal, postileal and total tract digestion of N. In trial 1, corn, oats and sorghum were each fed with coastal Bermuda grass hay in 75:25 ratios. Apparent prececal digestibilities were similar (P>.05) and averaged 46.6%. By-difference procedures were employed to calculate digestibility of the cereal grain N only and apparent prececal N digestibility averaged 57.1%. In trial 2, a basal corn and hay diet was supplemented with cottonseed meal and soybean meal. Apparent total tract N digestibilities were similar (P>.05) across treatments, and prececal digestibility averaged 45.6%. By-difference calculations were used to determine digestibility of SBM and CSM N alone. Apparent prececal digestibility of SBM was 52.5% and was lower (P<.05) than 81.2% for CSM. It is possible that inadequate or excessive heat treatment of SBM affected enzymatic digestion. True digestibility of total rations fed in trial 2 was estimated by linear regression of nitrogen digested on nitrogen intake or N presented to the large intestine. True N digestibility of diets containing SBM and CSM was 54.7% and 69.4%, respectively.     

Ovulation rate and litter size in gilts immunized against androstenedione and 17alpha-hydroxyprogesterone

Two experiments were conducted to evaluate the effects of the immunization of gilts against ovarian steroids on ovulation rate and litter size. In Exp. 1, gilts (n = five gilts/treatment) at 165+/-1.6 d of age were immunized against either carrier (Control), androstenedione, or 17alpha-hydroxyprogesterone. Age at puberty and estrous cycle length averaged 208+/-5.5 (P = 0.67) and 20.3+/-2.8 d (P = 0.41), respectively, and were not affected by treatment. The androstenedione- and 17alpha-hydroxyprogesterone immunized gilts had higher (P < 0.02) ovulation rates than Controls (14.2, 14.2, and 11.4+/-0.8, respectively). Total pigs born (P = 0.66) and pigs born live (P = 0.65) for the androstenedione-treated group were not different from Controls. Gestation length was not different (P = 0.36) between any of the treatments and the Controls (115+/-0.9 d). Procedures used in Exp. 2 were similar to those in Exp. 1, except that only Control (n= 18) and 17alpha-hydroxyprogesterone (n = 16) treatments were included and only litter size at farrowing was measured. Total pigs and pigs born live were higher in the 17a-hydroxyprogesterone-treated gilts than in the Controls (12.6 vs 10.5+/-0.6, P < 0.02; and 11.4 vs 9.2+/-0.6; P < 0.01, respectively). Data from this study indicate that litter size in gilts can be increased by immunization against 17alpha-hydroxyprogesterone.     

Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

Influence of body condition and bovine somatotropin on estrous behavior, reproductive performance, and concentrations of serum somatotropin and plasma fatty acids in postpartum Brahman-influenced cows

Ninety-nine multiparous Brahman-influenced (1/4 to 3/8 Brahman) cows were managed to achieve low (BCS = 4.3 +/- 0.1; n = 50) or moderate (BCS = 6.1 +/- 0.1; n = 49) body condition (BC) to determine the influence of bovine somatotropin (bST) on estrous characteristics, reproductive performance, and concentrations of serum GH and plasma NEFA. Beginning 32 d postpartum, cows within each BC were assigned randomly to treatment with or without bST. Non-bST-treated cows received no treatment, and treated cows were administered bST (Posilac, 500 mg s.c.) on d -35, -21, and -7 before initiation of the breeding season. On d -7, all cows received an intravaginal, controlled internal drug-releasing (CIDR) device. On d 0 (initiation of the 70-d breeding season), the CIDR were removed and cows received prostaglandin F2alpha (PGF2alpha). Blood samples were collected from the median caudal vein of the cows at each bST treatment and at d -28 and 0. Estrous behavior was monitored by radiotelemetry during the first 30 d of the breeding season. Growth hormone was increased (P < 0.05) in low and moderate BC cows treated with bST. The percentage of cows detected in estrus during the first 30 d of the breeding season was decreased (P = 0.05) for low BC (64%) compared with moderate BC (82%) cows. The interval to first estrus tended (P = 0.07) to be shorter in low BC-bST-treated cows (3.7 +/- 1.9 d) than in moderate BC-bST-treated cows (9.6 +/- 1.8 d). During the first 30 d of the breeding season, cows in low BC had a decreased (P = 0.02) number of mounts received and increased (P = 0.001) quiescence between mounts compared with cows in moderate BC. The number of mounts received was reduced (P = 0.04) in bST-treated cows. More (P = 0.02) cows treated with bST became pregnant during the first 3 d of the breeding season compared with non-bST-treated cows. The cumulative first-service conception rate tended (P = 0.07) to be greater for bST-low BC cows than non-bST-treated cows in low or moderate BC. On d 0, NEFA were greater (P < 0.05) in bST-treated vs. non-bST-treated cows. Low BC and bST reduced the intensity of behavioral estrus in postpartum Brahman-influenced cows. However, bST increased the first-service conception rate during the first 30 d of breeding and pregnancy rates during the first 3 d of breeding in postpartum Brahman-influenced cows.     

Nitrogen metabolism in mature horses at varying levels of work

Nitrogen metabolism was measured in five mature geldings performing varying levels of work that simulated race training. Following an initial maintenance period without forced exercise, workload was increased in succeeding 18-d periods by doubling the distance the horses were galloped in each period from period 2 through 4. A 4-d N balance experiment was conducted at the end of each 18-d period. The maximum distance galloped daily, which was in period 4, was repeated in period 5. Then, workloads were decreased through period 7. Nitrogen balance was determined in two additional periods, 8 and 9, while the geldings were at maintenance with no forced exercise. Geldings were fed diets containing similar protein-to-calorie ratios in all periods. Nitrogen retention increased (P less than .05) as workload increased from period 1 to period 3 and remained elevated throughout the rest of the experiment. Nitrogen retention did not (P greater than .05) decrease from highest values, observed at maximum workload during periods 4 and 5, until period 9, which was 34 d postexercise during a maintenance period. Nitrogen retention was higher (P less than .05) following training than before training.     

Physiological responses in foals weaned by abrupt or gradual methods

Physiological responses to weaning procedures were studied in 21 foals assigned to one of five treatments: (1) abrupt, total separation of mare and foal, no preweaning creep feed (TSNC); abrupt, total separation with preweaning creep feed (TSC); partial separation of mare and foal allowing fenceline contact, no preweaning creep feed (PSNC); (4) partial separation with creep feed (PSC); and (5) control (CON) no separation of mare and foal, foals creep fed. Changes in adrenal response to exogenous ACTH, basal and peak plasma cortisol concentrations, plasma triiodothyronine (T3) and thyroxine (T4) concentrations, weight gains and feed consumption were measured. Foals on the total separation treatments had higher adrenal responses (P<.05) and pre-ACTH basal (P<.05) and post-ACTH peak plasma cortisol concentrations (P<.05) than foals on other treatments indicating they were stressed at weaning. The PSNC, PSC and CON treatments did not differ (P>.05) in any cortisol response. No treatment differences were found in thyroid hormone concentrations in this study. On partial separation treatments, creep-fed and non-creep-fed foals consumed similar amounts of feed during the first week postweaning. On total separation treatments, non-creep-fed foals consumed more feed (P<.05) than creep-fed foals. All foals without creep feed gained more weight immediately after weaning (0–2 weeks) than creep-fed foals (P<.05), reflecting higher feed intakes and possible compensatory gains. Total postweaning weight gains (0–8 weeks) of foals were not significantly affected by treatment.