Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Flow cytometric sexing of X- and Y-chromosome-bearing sperm in Sika deer (Cervus nippon)

The objectives of the study were to determine a practical method of using predetermined sexed semen in Sika deer (Cervus nippon). Semen was collected by electro-ejaculation from two Sika stags and transported to the laboratory and separated into X- and Y-chromosome-bearing sperm after analysis and re-analysis (using a modified high-speed cell sorter), or control (unsorted) semen. Eighty-four Sika hinds were inseminated with 2.8 × 10⁷ unsorted (control) or 2.3 × 10⁶ sorted (X or Y) frozen-thawed semen via intra-uterine laparoscopy 58–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and the administration of 330 IU PMSG at the time of CIDR removal. No significant differences in the post-thaw motility of control (43.4 ± 4.4%), X- (45.3 ± 4.5%) and Y-sorted (43.5 ± 3.2%) samples were recorded. The sorted frozen-thawed sperm (X, 72.5 ± 6.4%: Y, 75.2 ± 5.5%) recorded significantly (P < 0.05) more intact acrosomes following thawing than the unsorted frozen-thawed (68.2 ± 10.2%) sperm. The individual Sika stags had no effect on the post-thaw sperm motility. Sorted frozen-thawed sperm demonstrated a significantly shorter survival time after thawing than the control sperm (P < 0.05). The number of Sika hinds pregnant following insemination with unsorted or control thawed sperm was significantly higher (33/42; 78.6%) than for hinds inseminated with either X- (5/11; 45.5%) or Y-sorted sperm (15/31; 48.4%). Ultimately 14 out of the 15 calves produced by Sika hinds inseminated with Y-sorted sperm were male (92.9%) and 5/5 calves (100%) from Sika hinds inseminated with X-sorted sperm were female. The sex ratio of the calves born to hinds inseminated with sex-sorted sperm significantly (P < 0.05) deviated for the 48.5% (female, 16/33) and 51.5% (male, 17/33) in the control group. All calves were born between 230 d and 243 d of gestation. Male and female calves in the control group had similar birth and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal calves of the predicted sex may be produced after intra-uterine insemination conducted by laparoscopy with low numbers of sex-sorted cryopreserved Sika sperm.     

Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy

The goal of the present study was to characterize the semen quality of dogs naturally infected with Leishmania chagasi, and treated with Allopurinol and Amphotericin B. Eight naturally infected and eight non-infected dogs were selected. Following semen collection, progressive motility, vigor, concentration and sperm morphology were evaluated. The seminal patterns in the treated animals were evaluated at the beginning (d0) and at days 30 (d30), 60 (d60) and 150 (d150) of treatment. The progressive motility at d0 (35.7 ± 22.3%) was less than that of the control group (77.8 ± 7.1%) (P < 0.05). The vigor was similar to the control group throughout the treatment (P > 0.05). The number of sperm/mL, sperm/ejaculate and sperm/kg of body weight was similar among groups (P > 0.05). The percentages of normal spermatozoa of infected and treated animals were similar throughout the treatment and to the control group (69.1 ± 8.7%) at d60 (37.5 ± 11.2%) and d150 (48.3 ± 10.8%) (P > 0.05), but smaller at d0 (22.7 ± 10.5%) and d30 (28.8 ± 15.9%) (P < 0.05). A greater percentage of acrosome damage was observed in the control group (3.1 ± 2.3%) compared to the d60 (0.1 ± 0.2%) (P < 0.05). The infected dogs had a greater percentage of principal piece defects at d60 (37.0 ± 6.3%) than the control group (16.8 ± 7.3%) (P < 0.05); and greater percentages of detached normal heads at d0 (28.7 ± 19.7%) and d30 (18.5 ± 18.5%) than the control group (0.4 ± 0.5%) (P < 0.05). This reduction in semen quality of the infected animals is suggestive of an epididymal dysfunction. Due to this poor semen quality, caution is recommended when using infected male dogs for reproductive purposes.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Sperm loss using different artificial vaginas in rams

The objective of the present study was to evaluate the effect of certain factors on sperm loss during ram semen collection, using an artificial vagina (AV). The factors analyzed were the effect of the male (8 rams), length of the artificial vagina (short versus long), order of the ejaculate (first versus second ejaculate) and lubrication technique (with or without lube). An observational study from a data set containing 55 semen collections from the 8 rams of different breeds (Montadale, Suffolk, Hamphshire, Polypay) were evaluated during the breeding season using a multiple regression statistical analysis over a period of 10 weeks. The results did not show any differences between rams, order of the ejaculate or the use of lubrication on sperm loss. The two types of artificial vaginas however showed significant differences in the percentage sperm loss (P < 0.001). The total ejaculate volume (collection tube volume + liner and cone recovery volume) and sperm concentration/ml (×10⁹) were similar when using the two types of artificial vaginas. The total ejaculate volume recorded for the short artificial vagina was 1.5 ± 0.4 ml and for the long artificial vagina was 1.3 ± 0.6 ml. The concentration of the ejaculate was 2.7 ± 0.6 × 10⁹ sperm/ml for the short artificial vagina and 2.9 ± 0.7 × 10⁹ sperm/ml for the long artificial vagina. The volume of semen in the collection tube using the short artificial vagina was 1.3 ± 0.4 ml, compared to the 0.7 ± 0.5 ml for the long artificial vagina (P < 0.001). The percentage of sperm loss from the short artificial vagina (12.9 ± 5.9%) was significantly lower than when using the long artificial vagina (50.8 ± 13.9%; P < 0.001). From this study, it may be concluded that the type of artificial vagina affects the sperm loss; with the shorter AV recording a lower sperm loss. No effects were detected between rams, order of the ejaculate or the use of lubrication on sperm loss. From the results obtained the use of the short artificial vagina can be recommended.     

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22 h in the presence of two doses (“low” and “high”) of androstenedione (A₄) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A₄: 337.5 and 562.5 ng/ml; T: 22.2 and 42.6 ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0 ± 2.1%) than any other combination of treatments (plasticware × A₄: 71.5 ± 2.6%; glassware × T: 71.2 ± 1.9%; and glassware × A₄: 71.4 ± 2.4%). The blastocyst formation rate for the plasticware × T treatment (39.7 ± 2.5%) was significantly greater than for all other combinations (glassware × T: 28.7 ± 2.2%; glassware × A₄: 24.0 ± 2.8%; and plasticware × A₄: 19.8 ± 3.0%) and the low dose of T (37.1 ± 2.5%) resulted in higher (p < 0.05) blastocyst formation rates than all other treatments (T high dose: 29.2 ± 2.5%; A₄ high dose: 27.1 ± 2.9%; and A₄ low dose: 20.2 ± 3.0%). The proportion of male embryos was greater (p < 0.05) in plastic than glass dishes in the low-dose A₄ group (59.1 ± 8.7% vs. 38.2 ± 5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p < 0.08) in the control groups and high-dose A₄ group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r = 0.38, p < 0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.     

Protective effect of taurine,glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h (P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h (P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C (P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.     

Effects of dietary conjugated linoleic acid on metabolic status,BW and expression of genes related to lipid metabolism in adipose tissue of dairy cows during peripartum

Conjugated linoleic acid (CLA) dietary supplementation reduces milk fat content and yield, but its effects on lipid metabolism and energy status remain controversial. The objective of this study was to investigate the effects of dietary CLA on adipose tissue (AT) mRNA abundance of genes related to lipid metabolism, plasma indicators of metabolic status, body condition score (BCS) and BW changes in dairy cows. Sixteen multiparous Holstein cows (3.2 ± 1.4 lactations, 615 ± 15 kg BW) were randomly assigned to treatments: 1) CLA; rumen-protected CLA (75 g/d) or 2) Control; equivalent amount of rumen inert fatty acid (FA) as the previous diet (78 g/d), from − 20.2 ± 3.2 (mean ± SEM) to 21 d relative to calving (d 0). Subcutaneous AT was biopsied from the tail-head region at d 21 to determine the mRNA abundance of genes related to lipid metabolism. Blood samples were collected at − 20.2 ± 3.2, 0, 7, 14 and 21 d relative to calving to determine plasma non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), insulin and glucose. Conjugated linoleic acid decreased milk fat yield and milk fat content by 15 and 16%, respectively. Cows fed CLA had lower plasma NEFA and BHBA and greater glucose and insulin concentrations (P < 0.05). Mean BCS at 21 d postpartum was greater (P < 0.01; 2.89 vs 2.25), and BCS loss from the day of enrollment to 21 d postpartum was reduced (P < 0.01; − 0.13 vs − 0.64) in the CLA group. The expression of acylcoenzyme A oxidase, carnitine palmitoyltransferase 1A, hormone-sensitive lipase, β2 adrenergic receptor and acetyl-CoA carboxylase was downregulated by CLA supplementation, whereas the expression of sterol regulatory element binding protein, lipoprotein lipase and peroxisome proliferator-activated receptor gamma was upregulated (P < 0.01). In summary, CLA-supplemented cows showed signs of better metabolic status and less severe fat mobilization. Moreover, CLA increased mRNA abundance of genes related to lipogenesis and decreased mRNA abundance of genes related to FA oxidation and lipolysis in the AT of dairy cows during early lactation.     

Effect of dietary β-glucan on growth performance,fecal microbial shedding and immunological responses after lipopolysaccharide challenge in weaned pigs

Two experiments (Exp.) were conducted to evaluate the effects of β-glucan inclusion in the diet on growth performance and immune function after lipopolysaccharide (LPS) challenge. In Exp. 1, a total of 40 weaned pigs (progeny of Landrace×Yorkshire sows by Duroc) with an initial body weight (BW) of 7.89 ± 0.84 kg (21 ± 2 d) of age) were used in a 28-day (d) experiment to determine the effects of dietary β-glucan on growth performance. Pigs were allotted randomly to two treatments consisting of addition of 0 or 0.1 g β-glucan/kg diet with four replicate pens per treatment and five pigs per pen. Growth performance was not affected by β-glucan supplementation throughout the experiment. However, dietary β-glucan reduced (P<0.05) the number of fecal Escherichia coli. In Exp. 2, a total of 20 weaned barrows (6.22 ± 0.25 kg of BW and 21 ± 2 d of age) individually raised in metabolic cages were used to evaluate immunological responses following LPS challenge. Pigs were fed 0 or 0.1 g β-glucan/kg diet for 42 d. At the end of the trial, half of the pigs (n = 5) from each treatment were injected intraperitoneal with E. coli LPS at a concentration of 100 μg/kg BW and the other half were injected with sterile saline solution. Treatments were arranged as a 2×2 factorial, with the main effect of LPS challenge (saline vs. LPS) and β-glucan supplementation (0 g/kg vs. 0.1 g/kg). After LPS injection, blood was taken at 0, 2, 4, 6, 8 and 12 hours (h) for the blood cell counts and blood inflammatory response. Dietary β-glucan increased (P<0.05) leukocytes counts at 4, 6 and 8 h, and blood lymphocyte concentrations at 2, 4 and 6 h and LPS challenge increased (P<0.05) counts of leukocytes at 2, 4, 6 and 8 h and blood lymphocyte at 2 and 4 h post-challenge. The rectal temperature was increased (P<0.05) at 2, 4, 6 and 8 h after LPS challenge. Dietary β-glucan reduced (P<0.05) and LPS challenge increased (P<0.05) blood plasma tumor necrosis factor-α (TNF-α) concentration at 2 and 4 h post-challenge. Dietary β-glucan increased (P<0.05) the concentration of the cluster of differentiation antigens 4 cells (CD4⁺) at 2, 4 and 6 h, and of 8 (CD8⁺) at 4 and 6 h post-challenge, respectively. The LPS challenge increased (P<0.05) CD4⁺ and CD8⁺ cell concentrations at 2, 4 and 6 h post-challenge. The CD4⁺:CD8⁺ ratio was reduced (P<0.05) by LPS challenge but was increased (P<0.05) by dietary β-glucan at 2, 4, 6 and 8 h post-challenge. In conclusion, dietary β-glucan decreased E. coli numbers but did not affect growth performance in weaned pigs and may offer benefits on immune function in weaned pigs challenged with LPS.     

Effect of individual and mixed live yeast culture feeding on growth performance,nutrient utilization and microbial crude protein synthesis in lambs

This study investigated effects of feeding three individual, and a mixed, yeast culture (Kluyveromyces marximanus NRRL3234, Saccharomyces cerevisiae NCDC42, Saccharomyces uvarum ATCC9080 all in a 1:1:1, ratio) on growth performance, nutrient utilization and microbial crude protein (CP) synthesis in feedlot lambs during the post-weaning phase of growth. Sixty weaner lambs (90 ± 3.5 d old and 15.9 ± 0.50 kg BW) were fed for 91 d in five equal groups. The control group of lambs received sterilized culture medium while the treatment groups were fed a yeast culture in addition to a ad libitum total mixed ration (TMR). The yeast culture, dosed at 1 ml/kg body weight (BW) had 1.5–2.0 × 10⁹ live cells/ml. Yeast culture supplementation did not influence intake and digestibility of organic matter (OM), CP, neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose and the metabolizable energy (ME) level of the diets were similar between control and yeast supplemented lambs. Lambs in all groups were in positive N balance, but N intake and N voided in feces and urine, as well as N balance, did not change due to yeast culture supplementation. Urinary allantoin excretion was similar, but purine derivatives absorbed (mM/d) were higher (P<0.05) in yeast culture supplemented lambs. Yeast culture supplementation improved (P<0.05) microbial CP synthesis. Supplementation of SC and mixed yeast improved (P=0.002) BW gain of lambs by 21% and 16% respectively. All yeast culture supplemented lambs had higher feed efficiency in comparison to control lambs. Among the three yeast cultures used, S. cerevisiae had the most potential as a growth promoting feed additive in feedlot lamb production, and it may serve as an alternate to antibiotics and ionophores as a growth promoter of weaner lambs.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Betacarotene supplementation increases ovulation rate without an increment in LH secretion in cyclic goats

This study aimed to evaluate the effects of betacarotene (BC) supplementation on ovulation rate (OR) and luteinizing hormone (LH) secretion in adult goats during the breeding season. Additionally, total ovarian activity (TOA) comprising the total number of ultrasonographically detectable antral follicles (AF) and corpora lutea (OR) was also assessed. In early October, adult goats [n = 22, 3.5 years of age, 7/8 Sannen-Alpine; 26°N, 103°W at 1117 m.a.s.l.] were randomly assigned to: (i) BC group (BCG), orally supplemented with 50 mg of BC/goat/day [n = 10; live weight (LW) = 45.9 ± 2.0 kg, body condition score (BCS; range: 0-emaciated to 5-obese) = 3.0 ± 0.1], and (ii) control group (CONT) [n = 12; LW = 46.2 ± 2.0 kg, BCS = 3.0 ± 0.1]. All animals received a basal diet of alfalfa hay, corn silage and corn grain, with free access to water and mineral salts. The whole experimental period spanned 34 days before and 17 days after ovulation. On day 23 of the experiment, estrus was synchronized with progestin-releasing intravaginal sponges; 36 h prior to estrus, an intensive blood sampling (every 15 min for 6 h) was performed to determine mean LH concentrations, pulsatility (LH-PULSE) and area under the curve (LH-AUC) for serial LH concentrations. Afterwards, by the end of the luteal phase (i.e., 17 days after the onset of estrus), an ultrasonographic scanning was performed to evaluate OR and TOA [AF + OR]. The average LW and BCS did not differ (p > 0.05) during the experimental period. BC-supplemented goats showed an increase in OR (3.4 ± 0.2 versus 2.8 ± 0.2; p < 0.05) and exhibited lower (p < 0.05) serum LH concentrations, LH-AUC and LH-PULSE compared to CONT. A positive correlation was recorded between OR and LW (r² = 0.42, p < 0.05) and BCS (r² = 0.47, p < 0.05). In addition, AF (5.0 ± 0.6 versus 3.4 ± 0.6) and TOA (8.4 ± 0.6 versus 6.2 ± 0.6) were greater (p < 0.05) in the BC-supplemented group than CONT. Supplementation with BC enhanced ovarian follicular development and ovulation rate in adult female goats under decreased photoperiods through LHRH-independant pathways or direct effects of BC on ovarian function.     

Time-lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope™ time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9 ± 0.1 h (range 0.70–10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p < 0.05) with shrinkage ceasing after 2 h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01) or 35–38 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01), but timing was not related to the Day 3 morphological grades (1–4) of subsequent embryos (2.9 ± 0.1, 2.9 ± 0.1, 2.8 ± 0.2 and 3.0 ± 0.1; p > 0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC = 0.596 or 0.601, p = 0.006 or 0.004) and usable embryos (AUC = 0.638 or 0.632, p = 0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Effect of high tannin grain sorghum on gastrointestinal parasite fecal egg counts in goats

The objective of three experiments was to determine the influence of high condensed tannin (CT) grain sorghum on gastrointestinal parasite fecal egg counts (FEC). Sixteen naturally infected Boer crossbred mixed-sex goats were used. Animals that were supplemented with grain daily were removed from pasture and placed in individual pens and fed treatment diets for 21 or 14 days (Experiment 3). Goats were allowed ad libitum access to water and diets containing high or low CT cracked grain sorghum. On day 0 and every 7 days thereafter, PCV, FEC and FAMACHA^® eyelid color scores (EYE; Experiment 1 only) were recorded. For Experiment 1, percentage of animals dewormed, PCV and EYE were not influenced by treatment and averaged 13.6 ± 4.5% per treatment period, 23.4 ± 0.8% and 3.2 ± 0.12, respectively, for all animals. The FEC increased after day 0 for control but not high tannin grain sorghum fed goats (treatment by day interaction, P < 0.02). As expected, EYE and PCV were negatively related (r = −0.45; P < 0.0005). For Experiment 2, there was no influence of diet on PCV or FEC, but there was an effect of day on FEC (P < 0.002) in which all goats had higher FEC on day 0 (1956 ± 219 eggs/g) than any other sampling day. For Experiment 3, there was no effect of feeding high CT grain sorghum on FEC (2992 ± 591 eggs/g) or PCV (25.1 ± 0.5%). In these studies, high CT grain sorghum did not consistently influence FEC and did not impact PCV or number of animals requiring chemical anthelmintic treatment.     

Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

Serum progesterone concentrations,follicular development and time of ovulation using a new progesterone releasing device (DICO®) in sheep

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO^®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO^® (n = 9) and CIDR-G^® (n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO^® (n = 8) and CIDR-G^® (n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO^® and CIDR-G^® devices, respectively. In Experiment 3, the re-utilization of DICO^® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO^® (n = 11) for a period of 6 days. The re-used DICO^® devices induced a lower serum progesterone concentration than the new devices (P < 0.05). However, the re-used DICO^® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO^® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO^® devices for 6 days was applied with (n = 8) or without (n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO^® or CIDR-G^® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.