Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day = D0), zygotes were cultured on granulosa cells + M199 + 10% serum + 100 μM GSH supplemented with 100 μM of t10, c12 CLA (CLA group, n = 1394) or without supplementation (control group, n = 1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n = 24; group B-CLA, n = 23). Non-biopsied embryos were also frozen (group NB-Control, n = 49; group NB-CLA, n = 45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24 h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P ≤ 0.003) and a smaller (P < 0.001) fat embryo index being leaner (P = 0.008) than control embryos. Embryo postwarming survival was higher in B-CLA than in B-control group (95.0 ± 7.0% versus 62.5 ± 7.9%; P < 0.001). After 24 h of culture, the viability (expansion rate) of biopsied embryos and nonbiopsied embryos, cultured with t10, c12 CLA was higher than control embryos (B-CLA = 64.6 ± 4.4% and B-control = 27.5 ± 2.5%, P = 0.01; NB-CLA = 86.0 ± 3.5% and NB-Control = 68.6 ± 7.0%, P = 0.05). Results showed that supplying t10, c12 CLA to serum-containing media decreases embryo cytoplasmic lipid deposition during in vitro culture and significantly improves resistance of IVP embryos to micromanipulation and cryopreservation.     

Glucocorticoids suppress vasopressin gene expression in human suprachiasmatic nucleus

Sleep impairment is one of the major side effects of glucocorticoid therapy. The mechanism responsible for this circadian disorder is unknown, but alterations in the suprachiasmatic nucleus (SCN), the biological clock of the human brain, are presumed to play a major role. In the present study, the amount of vasopressin mRNA (AVP mRNA) expression in the SCN was investigated in 10 glucocorticoid-exposed patients and 10 glucocorticoid free, age- and clock time of death-matched controls. The total amount of AVP mRNA, expressed as masked silver grains in the SCN, was two times lower in glucocorticoid-exposed patients (n = 10; 5115 ± 1314 μm²) than that in controls (n = 10; 11,021 ± 1408 μm²) (P = 0.006). There was also a 53% decrease in the total number of profiles in the SCN that expressed AVP mRNA in glucocorticoid-exposed patients (16,759 ± 3110) compared with those in controls (31,490 ± 3816) (P = 0.01). In conclusion, glucocorticoids have an inhibitory effect on AVP mRNA expression in the human SCN, which may be the biological basis of the circadian rhythm disturbances during glucocorticoid therapy.     

Control of postpartum anestrous with an intra-vaginal progesterone device plus eCG or calf removal for 120 h in suckled crossbred cows managed in a pasture-based system

To control postpartum anestrus and reduce calving to conception interval, 167 crossbred non-pregnant cows that were 90–130 days postpartum were allotted randomly to one of the following treatments: PH (n = 59), intra-vaginal sponge with 250 mg of medroxyprogesterone acetate (MAP) for 7 days plus 50 mg of MAP and 5 mg 17-β estradiol (17β-E) in the first day of treatment (day −8), 500 UI eCG (day −3) and 1.5 mg 17β-E in 24 h after sponge removal (day 0); CR (n = 57), temporary calf removal for 120 h; CG (n = 51), control group without treatment. Estrus rate differed among treatments (P < 0.01) being greater in PH (78.2%), followed by CR (52.0%) and CG (22.9%). A greater proportion of cows in the PH (80.0%) and CR (54%) groups had ovulations when compared to CG (35.4%). Intervals to first estrus were 13.5 ± 6.3 days, 26.1 ± 6.4 days and 52.5 ± 7.5 days for the PH, CR and CG groups, respectively. First insemination conception was similar in the three groups. Postpartum intervals to first breeding (PFS) and to conception (PCI) were longer in CG than PH and CR groups (P < 0.05; P < 0.01). The PH and CR groups had a similar PFS but PCI was different (P < 0.02). Accumulated pregnancy rate at 30 and 60 but not at 90 days were different (30 days: P < 0.09; P < 0.01; P < 0.09; 60 days: P < 0.06; P < 0.01; P < 0.03) among treatments. After 90 days post-treatment, 9%, 18% and 33% of cows from the PH, CR and CG groups had not conceived. Similarly, 5.4%, 6.0% and 12.5% of cows from the PH, CR and CG groups, respectively, were culled from the herd because of lack of pregnancy after 180 days post treatment. In the group of cows evaluated by ultrasonography, only those cows having larger ovaries and dominant follicles had ovulations. It was concluded that the hormonal treatment was more efficient in inducing a fertile estrus and reducing calving to conception interval followed by the calf removal for 120 h. Each method can be considered as an important tool to reduce the postpartum anestrous period in dual purpose herds when AI is conduct in the tropics.     

Serum concentrations of Cu, Zn, Fe, Mo and Co in newborn lambs following systemic administration of Vitamin E and selenium to the pregnant ewes

This study was conducted to determine the effects of systemic administration of Vitamin E and selenium to pregnant ewes at the late stage of gestation on serum concentrations of Cu, Zn, Fe, Mo and Co in their offspring's. Pregnant Lori–Bakhtiari ewes (n = 14) were randomly assigned to receive Vitamin E and selenium (treatment group; n = 7) or distilled water (control group; n = 7), once 3 weeks and again 1 week before parturition. Blood samples were taken from jugular vein of lambs at parturition and once a week during the 4 week of age. Serum concentration of Fe, Cu, Zn, Mo and Co was determined by atomic absorption spectrophotometery.

At parturition, serum concentration of Cu, Zn, Mo and Co were identical in lambs of both groups, while the serum concentration of Fe (mean ± S.E.) was significantly higher in lambs of control (210.83 ± 9.05 μg/dl) than treatment group (140.71 ± 17.8 μg/dl).

From parturition to the forth weeks of age the serum concentrations of Fe and Cu were increased (P < 0.05) in lambs of treated group which was concomitant with a reduction in Zn concentration.

In conclusion, increase in serum concentrations of Cu and Fe during the first 4 weeks of age in lambs of ewes given vitamin E and selenium compound, could disturb the Zn:Cu and Zn:Fe ratios which in turn lead to zinc deficiency.     

Body condition and protein supplementation positively affect periovulatory ovarian activity by non LH-mediated pathways in goats

Effects of rumen undegradable intake protein (UIP) supplementation on ovarian activity and serum insulin, GH, and LH were evaluated in goats having low or high body condition (BC). Goats with either low BC (n = 16, 28.7 ± 0.8 kg BW, BC = 2.1 ± 0.3) or high BC (n = 16, 38.4 ± 0.8 kg, BC = 3.2 ± 0.3) received, during 40-days, one of the two protein supplementation levels: without UIP or with UIP (120 g goat⁻¹ d⁻¹). Oestrus was synchronized with two i.m. doses of PGF₂, and jugular blood samples were collected from 36 to 42 h after the second prostaglandin injection at 15 min intervals. Serum concentrations of insulin, LH, and GH were measured The number of preovulatory follicles and the number of corpora lutea (CL) were evaluated by transrectal ultrasonography at 1 and 4 days after the second prostaglandin dose, respectively. Does with higher BC had more CL than those in the lower condition group (2.8 ± 0.2 versus 1.8 ± 0.2, P < 0.05). Similarly, goats receiving UIP supplementation had more follicles (2.6 ± 0.2 versus 1.9 ± 0.2, P < 0.05) and tended to have more CL (2.6 ± 0.2 versus 2.0 ± 0.2, P = 0.05) than does not receiving UIP. Neither BCS nor UIP supplementation affected serum GH or LH concentrations, pulsatility, or area under the curve. High BC does produced more insulin (1.92 ± 0.17 versus 0.81 ± 0.17 ng/mL, P < 0.01 ng/mL) than lower BC goats; the same for UIP-supplemented (1.69 ± 0.18 versus 1.04 ± 0.18, P < 0.05). Results suggest that the increased ovarian activity observed in both UIP-supplemented and higher BC goats was not the result of changes in LH or GH, suggesting effects at a local level, through changes in insulin in a non-GnRH-gonadotrophin dependent manner.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Parity of female goats does not influence their estrous and ovulatory responses to the male effect

The objective of the present study was to determine whether parity is a factor that influences the estrous and ovulatory responses of female goats when they are stimulated by males that show increased sexual activity. To stimulate sexual activity, four adult male goats were subjected to photoperiodic treatment for 2.5 months comprising long days, with the treatment commencing on 1 November. On 14 April at 1900 h, a group of multiparous females (n = 21) and a group of 16 months-old nulliparous females (n = 19) were exposed to four bucks (two per group) for 15 days. Throughout the study period, the estrous behavior of these female goats was detected twice on a daily basis. Ovulations of the female goats were determined by ecography on days 7 and 18 after exposure to males. The sexual behavior of males was recorded twice every day from 0800 to 0900 h and from 1730 to 1830 h during the first 4 days after introduction in the pen of females. The total cumulative proportion of multiparous females that had ovulations (100%) and displayed estrous behavior (100%) during the 15 days of exposure to males did not differ (P > 0.05) from that of nulliparous females (100% and 95%, respectively). The interval between introduction of males and onset of estrous behavior did not differ (P > 0.05) between multiparous (1.9 ± 0.1 days) and nulliparous (1.7 ± 0.2 days) females. The proportion of females displaying a short estrous cycle was greater (P < 0.05) in multiparous (13/21, 62%) than in nulliparous (5/19, 26%) females. Duration of these shorter than typical estrous cycles did not differ (P > 0.05) between groups (multiparous: 5.2 ± 0.3 days, nulliparous: 4.5 ± 0.1 days). The number of anogenital sniffings was greater (P < 0.001) in males exposed to nulliparous than in those exposed to multiparous females. In contrast, the number of mounting attempts was greater (P < 0.01) in males that were introduced to multiparous than in those that were introduced to nulliparous does. The number of flehmen, nudging, self-marking with urine, and mounts was not different (P > 0.05) between males that were in contact with multiparous and nulliparous females. These results indicate that regardless of parity, female goats respond to male introduction if they are stimulated by males that were previously exposed to artificial long days to increase their sexual behavior.     

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n = 83) were synchronized and the number of follicles (≥2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (±S.D.) (≥2 mm diameter) of follicles per ewe was 8.5 ± 2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n = 21) had fewer (P < 0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: ≥8 follicles/day; n = 23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68 ± 0.11 versus High 0.79 ± 0.08; P = 0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (≥8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2–3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

The effects of doxycycline administration on amino acid neurotransmitters in an animal model of neonatal hypoxia-ischemia

Neonatal hypoxia-ischemia (HI) is a major contributor to many neurological, psychiatric and behavioral disorders. Previous studies in our laboratory have shown that a one-time dose of doxycycline (DOXY), even when given 3 h after HI insult, was neuroprotective and significantly reduced microglial activation and cleaved caspase-3 protein expression in the immature brain. In light of these data, the goal of this study was to investigate the effects of DOXY administration on amino acid neurotransmitters. Post-natal-day 7 rats received DOXY (10 mg/kg) or vehicle (VEH) concomitant with the onset of HI, and were euthanized 30 min, 1, 2 or 4 h post-HI (n ≥ 6). Extracted brains were either immediately dissected for frontal cortex, striatum and hippocampal regions, or removed in their entirety and flash frozen in isopentane for histological analyses. Dissected regions were homogenized and aliquots were prepared for high performance liquid chromatography (HPLC) analyses of amino acid levels and brain levels of DOXY. HPLC extraction revealed that systemic administration of DOXY resulted in mean drug levels of 867.1 ± 376.1 ng/g of brain tissue. Histological analyses revealed microglial activation, caspase-3 activation and neuronal degeneration consistent with a mild injury in the regions most vulnerable to HI. We found that HI caused significant, time-dependent, regional changes in brain amino acids including glutamate, GABA, alanine, aspartate, asparagine, serine, glutamine, glycine and taurine. HI significantly increased glutamate levels in the hippocampus (HI + VEH = 15.8 ± 3.1 ng/μg versus control = 11.8 ± 1.4 ng/μg protein) 4 h post-HI (p < 0.05). Pups treated with DOXY had lower glutamate levels (13.1 ± 2.4 ng/μg) when compared to VEH-treated pups (15.8 ± 3.1 ng/μg), however these values failed to reach significance. In addition, DOXY-treated pups had significantly lower alanine (HI + VEH = 1.1 ± 0.2 ng/μg versus HI + DOXY = 0.5 + 0.1 ng/μg) and serine (HI + VEH = 1.4 ± 0.4 ng/μg versus HI + DOXY = 0.7 + 0.1 ng/μg) levels in the hippocampus, 4 h post-HI. Similar normalizations and significant reductions in alanine and serine were seen in the cortex and striatum. These results show that in addition to its previously reported and well-documented anti-inflammatory and anti-apoptotic properties, DOXY has significant effects on amino acid neurotransmitters.     

Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes

We evaluated chromosomal aberrations in lymphocytes of 177 workers exposed to xenobiotics in a tire plant and in 172 controls, in relation to their genetic background. Nine polymorphisms in genes encoding biotransformation enzymes and nine polymorphisms in genes involved in main DNA repair pathways were investigated for possible modulation of chromosomal damage. Chromosomal aberration frequencies were the highest among exposed smokers and the lowest in non-smoking unexposed individuals (2.5 ± 1.8% vs. 1.7 ± 1.2%, respectively). The differences between groups (ANOVA) were borderline significant (F = 2.6, P = 0.055). Chromosomal aberrations were higher in subjects with GSTT1-null (2.4 ± 1.7%) than in those with GSTT1-plus genotype (1.8 ± 1.4%; F = 7.2, P = 0.008). Considering individual groups, this association was significant in smoking exposed workers (F = 4.4, P = 0.040). Individuals with low activity EPHX1 genotype exhibited significantly higher chromosomal aberrations (2.3 ± 1.6%) in comparison with those bearing medium (1.7 ± 1.2%) and high activity genotype (1.5 ± 1.2%; F = 4.7, P = 0.010). Both chromatid- and chromosome-type aberration frequencies were mainly affected by exposure and smoking status. Binary logistic regression analysis revealed that frequencies of chromatid-type aberrations were modulated by NBS1 Glu185Gln (OR 4.26, 95%CI 1.38–13.14, P = 0.012), and to a moderate extent, by XPD Lys751Gln (OR 0.16, 95%CI 0.02–1.25, P = 0.081) polymorphisms. Chromosome-type aberrations were lowest in individuals bearing the EPHX1 genotype conferring the high activity (OR 0.38, 95%CI 0.15–0.98, P = 0.045). Present results show that exposed individuals in the tire production, who smoke, exhibit higher chromosomal aberrations frequencies, and the extent of chromosomal damage may additionally be modified by relevant polymorphisms.     

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.     

Behaviour during the first stage of labour in cattle: Influence of parity and dystocia

The aim of the study was to examine typical behavioural elements in suckling cows and heifers at the first stage of labour and how these are affected by parity and dystocia. For this purpose, the parturitions of 87 cattle (10 Simmental heifers; 77 multiparous cows, 55 Simmental, 21 Simmental × Limousin) were observed from the appearance of the amniotic sacs in the cervix up to the emergence of the foetal phalanges in the rima vulvae. The animals were divided into three groups: group 1—cows with eutocia, n = 68; group 2—heifers with eutocia, n = 10; group 3—cows with dystocia, n = 9.

Compared to cows, there was a lower proportion of heifers with calm behaviour, whereas a higher proportion of heifers showed pawing with the forefeet (P < 0.05).

There was a higher proportion of cows with dystocia which showed rubbing against the wall (P < 0.05), discharge of urine (P < 0.05) and scraping on the floor (P < 0.05) compared to cows with eutocia.

The differences in behaviour of cows and heifers should be considered in a system for monitoring parturition to avoid misconstruing the normal calving situation and to obviate unnecessary obstetric intervention in heifers. Behavioural elements which were more frequently detectable in cases of dystocia should draw attention to the possibility that there may be a problem in the first stage of labour.     

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly (P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly (P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly (P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential (r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant (P < 0.05) correlations with the mitochondrial membrane potential (r = 0.70) and plasmalemma integrity measured by HOS-G test (r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.     

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (− 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by − 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (− 8 ± 3 beats/min, n = 7). Combined blockade of NK₂ and NK₃ receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (− 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.     

Interaction of calf suckling, use of oxytocin and milk yield with reproductive performance of dairy buffaloes

Calf suckling and oxytocin injections are commonly used for pre-milking stimulus in dairy buffaloes under field conditions. A study was conducted to investigate effect of these treatments on reproductive performance. Fifty one Nili-Ravi buffaloes were monitored from parturition up to 150 days postpartum through rectal examination. Data on milk yield, body condition score (BCS) and reproductive parameters were recorded weekly. Postpartum ovulation interval (POI) was determined by presence of an ovulation depression or a very soft corpus luteum haemorrhagicum and was confirmed through milk progesterone levels (MPL). Suckling was used to stimulate milk let down, and where the calf had died, injection of oxytocin was resorted to. Milk samples were analyzed for MPL using radioimmunoassay (RIA) and fat; and milk yield was converted to 4% fat corrected milk (FCM). The mean postpartum uterine involution length (PUI) was 34.30 ± 1.33 days. Mean POI was 59.37 ± 4.76 days and mean postpartum estrus interval (PEI) was 69.03 ± 6.03 days. Suckling period averaged 26.40 ± 5.57 days and correlated with POI (r = 0.19, P < 0.01) and PEI (r = 0.23, P < 0.01). POI was shortest in buffaloes suckled for one month (P < 0.05). Oxytocin was used with a mean dosage of 7.50 IU, delaying placental expulsion time (PET) and POI but shortening PEI. BCS shortened PET, POI and PEI (P < 0.01). Mean FCM was 14.50 ± 0.20, ranging from 2 to 35 kg/d; and was higher in estrus group; correlating positively with POI (r = 0.31, P < 0.01). MPL were 1.37 ± 0.17 ng/ml and increased after ovulation, remaining greater than 1.5 ng/ml from Day 4 to 14 of the estrus cycle, followed by a rapid decline up to next estrus. BCS in buffaloes resuming oestrus was constantly higher than those failing to resume ovarian cyclicity. Live weight, prepartum was 510.0 ± 5.9 kg with a loss of 3.7 ± 2.12 kg, 30 days postpartum. The present study suggests a lower reproductive efficiency of dairy buffaloes under the peri-urban farming system reflected by ovarian cyclicity in 68.63% buffaloes within 150 days postpartum and silent estrus in 51.5% of the cases. Increasing suckling duration and use of oxytocin delayed POI, however, POI was shortest in buffaloes suckled for one month. The high yielding buffaloes also manifested better reproductive cyclicity; while moderate yielder showed shorter ovulation intervals and higher conception rate.     

Short term, repeated exposure to rams during the transition into the breeding season improves the synchrony of mating in the breeding season

The ram effect is widely used in Mediterranean breeds of sheep but its use in temperate genotypes is restricted by breed seasonality. However, ewes from these highly seasonal genotypes are sensitive to stimulation by rams close to the onset of the natural breeding season. In this study we developed a pre-mating protocol of repeated, short-term exposure to rams (fence-line contact or vasectomised rams) beginning during late anoestrus and continuing into the breeding season. We hypothesised that this pre-mating protocol would synchronise the distribution of mating of North of England Mule ewes during the breeding season above that observed in ewes isolated from rams prior to mating. Ram-exposed ewes were given contact with rams (Experiment 1: fence-line; FR, n = 94 and Experiment 2: vasectomised rams; VR; n = 103) for 24 h on Days 0 (10 September), 17 and 34 of the experiment. Control ewes (Experiment 1; FC, n = 98 and Experiment 2; VC; n = 106) remained isolated from rams prior to mating. In Experiment 2, a subset of VR (n = 35) and VC ewes (n = 35) were blood sampled twice weekly to monitor their pre-mating progesterone profiles. At mating, harnessed entire rams were introduced, 17 or 16 days after the last ram exposure (Experiments 1 and 2) and raddle marks were recorded daily. The median time from ram introduction to mating was reduced in ewes given both fence-line and vasectomised ram contact (P < 0.001), leading to a more compact distribution of mating and lambing (At least P < 0.01). In the blood sampled VR ewes, there was a progressive decline in the number of days from ram exposure to the onset of dioestrus (at least P < 0.05). This observation indicates that the cycles in VR ewes became increasingly synchronised over the pre-mating period, a pattern not evident in VC ewes. In conclusion, repeated, short-term exposure of ewes to rams during the transition into the breeding season is an effective method of synchronising the distribution of mating during the breeding season.     

Relationship between male-male and male-female sexual behavior in 5-6-month-old male lambs

To characterize male–male sexual behavior during lamb development, to relate it with lamb body and testicular growth, and with sexual behavior toward estrual ewes, 40 Milchschaf male lambs, weaned at 45 days of age, were kept with ewes that were nursing younger lambs. Experimental lambs were weighed and scrotal circumference was measured every 2 weeks. Male–male sexual behavior was observed during 1–2 h every 2 weeks after birth until 7 months of age. Observations were recorded more intensively (3–4 h on five different days) for 2 weeks (5–6 months of age) as male–male sexual behavior increased during that period. Both mounting and mounted lambs were identified. An individual mounting index (MI) was calculated. To study male–female sexual behavior, lambs were individually located with two estrual ewes, and during 5 min the number of ano-genital sniffing, lateral approaches, mounts, and mounts with ejaculation were recorded. From those data, a libido index was also calculated. Male–male mounts (n = 308) were observed. Courtship behavior was displayed in 25% of interactions; mounts were accepted in 72.1% of attempted mounts. Mounts without previous courtship were accepted more frequently than mounts with previous courtship (P = 0.002). Lamb weight and scrotal circumference were not different according to MI groups. Lambs that mounted more times estrual ewes (first tertile) had greater (P = 0.04) MI (0.61 ± 0.10) than lambs with medium (0.27 ± 0.09) and less (0.30 ± 0.10) MI. The regression between MI and heterosexual libido index was r = 0.33 (P < 0.05). In summary, intensive male–male sexual activity during a short period of male lamb development was observed. There was a positive relationship between sexual behavior of male lambs towards other male lambs and towards estrual ewes.     

Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation

The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P < 0.05) in the percentage of live, caspase-activated spermatozoa (2.9 ± 0.7% vs 14.2 ± 6.4%; mean ± S.E.M.), low mitochondrial membrane potential (MMP; 6.8 ± 1.1 vs 23.8 ± 3.7), altered plasma membrane permeability (1.3 ± 0.2 vs 3.0 ± 0.5), DNA fragmentation (2.0 ± 1.3 vs 14.3 ± 3.6), total motility (81.8 ± 3.3 vs 35.1 ± 5.4), and progressive motility (66.3 ± 4.3 vs 24.1 ± 4.5) for high-density versus low-density subpopulations, respectively. Phosphatidylserine externalization did not differ (P = 0.67) between the high- and low-density subpopulations (2.6 ± 0.7 vs 3.1 ± 0.9). After cryopreservation, equine spermatozoa differed (P < 0.01) in the percentage of active caspases (19.1 ± 1.6 vs 52.1 ± 2.8), low MMP (18.2 ± 2.5 vs 48.7 ± 2.6), altered plasma membrane permeability (6.8 ± 1.7 vs 17.6 ± 2.0), total motility (75.5 ± 2.4 vs 45.2 ± 5.6), and progressive motility (53.9 ± 3.1 vs 28.3 ± 4.5) for pre-freeze versus cryopreserved spermatozoa. There was no difference (P = 0.21) in percentage of DNA fragmented cells before (5.5 ± 1.2) versus after cryopreservation (6.6 ± 1.1). We concluded that apoptotic-like changes were detectable in ejaculated equine spermatozoa and were more prevalent after cryopreservation.     

Effects of Tasco (a seaweed extract) and heat stress on N metabolism and meat fatty acids in wether lambs fed hays containing endophyte-infected fescue

Wether lambs (n = 27, average BW = 40 kg) were used to test response to forage treated with Tasco-Forage (an extract of the brown kelp Ascophyllum nodosum) prior to conserving, or to direct feeding of the extract (Tasco-EX). Hays made from endophyte (Neoyphodium coenophialum)-infested tall fescue (Festuca arundinacea)-based pasture received 0 or 3 kg of Tasco/ha prior to harvest. Lambs, blocked by weight, were randomly allotted to three diets: (1) control hay, (2) treated hay, and (3) #1 + Tasco-EX fed at 1% of the diet. Hays were low in CP (<7%) so all lambs were fed soybean meal (12% of the diet) in addition to trace mineralized salt. Diets were fed at 1.5% BW to prevent refusals. Total collections (7 d) were made during periods without or with applied heat stress. After each period, rumen contents were obtained to determine pH, NH₃ and VFA. Lambs were sacrificed post-trial. A subset was used to evaluate sensory traits and muscle fatty acids. Lambs were in negative N balance during the study and Tasco treatments did not affect N metabolism. Fecal N tended (P < 0.10) to increase with short duration heat stress causing a concomitant decrease (P < 0.05) in apparent N digestibility (58.6 versus 56.1%; S.E. = 0.7). Urinary N loss decreased (P < 0.001) with heat stress (8.0 versus 5.9 g/d; S.E. = 0.2), resulting in increased (P < 0.001) N retention (−2.1 versus −0.3 g/d; S.E. = 0.2). Apparent OM digestibility was not affected by heat stress but was greater (P < 0.05) for lambs fed Tasco-EX treatment than those fed treated hay. Treatment diets decreased (P < 0.05) ruminal butyrate. Heat stress increased (P < 0.05) acetate and total VFA and decreased (P < 0.01) ruminal pH. A tendency (P < 0.11) of increased 14:1ω5, decreased (P < 0.05) 18:0 and total saturated fatty acids in muscle was observed with Tasco diets. Meat sensory characteristics were not affected by treatment. Tasco may alter some aspects of rumen or lipid metabolism but has no effect on N metabolism or meat sensory characteristics of sheep fed restricted, low-quality diets.