Theriogenology - Articles in Press

Short-term feed restriction decreases the systemic and intrafollicular concentrations of leptin and increases the vascularity of the preovulatory follicle in mares - Corrected Proof

M.O. Gastal, E.L. Gastal, M.A. Beg, O.J. Ginther — 2010-03-12

Abstract: The objective of this study was to evaluate the effect of short-term feed restriction on characteristics of the preovulatory follicle and on concentrations of systemic hormones (leptin, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and follicular fluid hormones and growth factors (leptin, estradiol, inhibin-A, activin-A, free insulin-like growth factor-1 [IGF1], insulin-like growth factor binding protein 2 [IGFBP2], vascular endothelial growth factor [VEGF]). Mares were submitted to a short-term (48 h) feed restriction when the expected ovulatory follicle was ≥27mm (Hour 0) or served as controls (n=8/group). No effect of short-term feed restriction was detected for systemic concentrations of FSH and LH and for intrafollicular concentrations of estradiol, activin-A, free IGF1, and IGFBP2. Restricted mares had decreased systemic concentrations of leptin at Hour 24 (approached significance) and at Hours 36 and 48 (P<0.04). Follicular fluid of restricted mares at Hour 48 had lower (P<0.02) concentration of leptin and a tendency (P<0.1) for greater concentrations of inhibin-A and VEGF. The percentage of wall of the preovulatory follicle with color-Doppler signals of blood flow at Hour 48 was greater (P<0.04) in the restricted group. Intrafollicular concentration of leptin (combined groups) was positively correlated with score for body condition (r=+0.60; P<0.002) and negatively correlated with the percentage of the follicle wall with blood-flow signals (r=−0.60; P<0.02). Our favored interpretation is that the preovulatory follicle seems to compensate for a nutritional deficiency by increasing the blood flow in the follicle wall.

Immunohistochemical visualization of insulin receptors in formalin-fixed bovine ovaries post mortem and in granulosa cells collected in vivo - Corrected Proof

2010-03-12

Abstract: Insulin is crucial for granulosa cell (GC) function, follicle growth and ovulation in cows; low insulin levels increase the risk for anoestrus. Apart from insulin concentration, alterations in the insulin receptor (IR) density on GC may affect follicular growth and steroidogenesis. Data about the IR protein distribution in the bovine follicle are scarce. Therefore, we aimed to develop a quantifiable staining method for IR protein on histological sections of bovine follicles in different developmental stages, and to apply this technique on GC obtained in living cows.In a first experiment, bovine ovaries were collected post mortem, formalin fixed, routinely processed, and stained with monoclonal murine IR-antibodies, peroxidase-labeled goat anti-mouse antibodies, and substrate chromogen. Based on their diameter, follicles were morphologically classified as small antral (SAF; n = 141), dominant (DF; n=28) or subordinate (SF; n=8); DF and SF were further classified as healthy or atretic based on the ratio of estrogen and progesterone concentrations in their follicular fluid. Using specialized software, the proportion of pixels displaying a positive staining signal was computed as a measure for IR density in three selected follicular regions: GC, theca (T) and stroma (STR). Results were analyzed in an ANOVA model with follicle type, region and health status as fixed factors. In SAF, DF, and SF, IR density was notably higher in GC than T or STR; the latter two displayed very low or no IR presence. The IR density in SAF was stronger than in DF and tended to be stronger than in SF. Staining intensity was not altered in atretic compared to healthy follicles. In corpus luteum, cumulus-oocyte complexes and pre-antral follicles, no IR could be detected. In a second experiment, GC samples were collected from 20 live cows on 30 and 70 d post partum by transvaginal follicular fluid aspiration, projected on glass slides, and stained using the protocol described above. Most samples yielded sufficient GC and IR was clearly visualized. However, objective quantification of the staining signal was impeded by extensive variation in the arrangement and density of GC and the amount of cellular debris on the slides.Altogether, strong IR presence in GC, most notably in SAF, suggests acquisition of IR as a key event in early follicle growth. Furthermore, we have developed a quantifiable staining technique for bovine follicles that may be applicable for GC obtained in live cows, although this method requires further standardization.

Reproductive performance of repeat breeders in dairy herds - Corrected Proof

2010-03-12

Abstract: The objectives were to characterize repeat breeding in dairy cows, including reproductive performance and risk factors. Data from 613 Holstein Friesian cows in nine dairy herds across Japan were enrolled. A repeat breeder was defined as a cow that did not become pregnant after three inseminations, despite no clinically detectable reproductive disorders. In contrast, cows that became pregnant within three inseminations were considered to have normal fertility. Of the 613 cows, 87.3% eventually became pregnant after repeated AI (maximum calving to conception interval was 435 d). Mean (±SEM) first AI conception rate, days in milk at first AI, calving to conception interval and service per conception were 38.3%, 82±2 d, 125±3 d, and 2.0±0.1 times, respectively. Normal fertility cows (n=479) required only 114±3 d to conceive and 1.7±0.1 inseminations per pregnancy, whereas repeat breeders (n=86) required significantly more days to conceive (211±10) and more inseminations per pregnancy (4.7±0.2). Based on survival analysis, it took 94 d after calving for 50% of normal fertility cows to become pregnant, compared to 155 d for repeat breeders. For repeat breeders, 31.4, 50.0, and 58.1% became pregnant within 210, 300, and 435 d after calving, respectively. The risk factors for repeat breeding were parity (relative risk [RR]=0.809; P=0.058), resumption of postpartum ovarian cycles (RR=1.928; P=0.009), and days in milk at first AI (RR=0.991; P=0.039). In conclusion, repeat breeder dairy cows had very poor reproductive performance. Lower parity, abnormal resumption of postpartum ovarian cycles, and shorter days in milk at first AI were risk factors for repeat breeding.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction - Corrected Proof

2010-03-11

Abstract: Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe2+) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe2+, EE-CAT plus Fe2+, EE-GPx plus Fe2+ and EE-SOD plus Fe2+). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe2+ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe2+ and the antioxidative enzymes supplementation plus Fe2+ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

In vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques - Corrected Proof

2010-03-11

Abstract: The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P<0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P<0.01), while CT sperm contained more spermatozoa with tail abnormalities (P<0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P>0.05) between CT and EP sperm. Nevertheless, no difference (P>0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.

Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay - Corrected Proof

R. Ghosal, R. Sukumar, P.B. Seshagiri — 2010-03-08

Abstract: Asian elephants (Elephas maximus), prominent “flagship species”, are listed under the category of endangered species (EN – A2c, ver. 3.1; IUCN Red List 2009) and there is a need for their conservation. This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal, ethical, and practical reasons. Hence, there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants, which will help in the species’ conservation strategies. In this study, we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e., allopregnanolone (5α-P-3OH) in fecal samples of Asian elephants. We validated the assay which had a sensitivity of 0.25μM at 90% binding with an EC50 value of 1.37μM. Using female elephants, kept under semi-captive conditions in the forest camps of Mudumalai Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India, we measured fecal progesterone-metabolite (5α-P-3OH) concentrations in six animals and showed their clear correlation with those of serum progesterone, measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P<0.1) between the profiles of fecal 5α-P-3OH (range: 0.5–10μg/g) and serum progesterone (range: 0.1–1.8 ng/mL). Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy.

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection - Corrected Proof

Candace C. Jacobson, Young-Ho Choi, Shelby S. Hayden, Katrin Hinrichs — 2010-03-08

Abstract: Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F2α was given on Days 6 and 7 after aspiration. A follicle ≥25mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.

Use of a five-day progesterone-based timed AI protocol to determine if flunixin meglumine improves pregnancy per timed AI in dairy heifers - Corrected Proof

2010-03-08

Abstract: Two experiments were conducted to test the hypothesis that the 5 d Co-Synch + CIDR (Controlled Internal Drug Release insert containing progesterone) protocol could be applied as an efficient timed AI (TAI) protocol in dairy heifers, and that treatment with flunixin meglumine (FM) during the period of CL maintenance would increase pregnancy per TAI (P/TAI) and late survival of embryos. Objectives were: 1) in Experiment 1, to compare P/TAI with the 5 d Co-Synch+CIDR protocol to a PGF2α/GnRH protocol; and 2) in Experiment 2, to determine if FM administered 15.5 and 16 d after first TAI would increase P/TAI, using the 5 d Co-Synch+CIDR protocol with a new or previously used (5 d) CIDR insert.In Experiment 1, 248 heifers were assigned randomly to either the PGF2α/GnRH protocol (n=120) or the 5 d Co-Synch+CIDR protocol (n=128). Pregnancy per TAI did not differ between the 5 d Co-Synch+CIDR protocol (53.1%) and the PGF2α/GnRH protocol (45.8%; P=0.22). In Experiment 2, 325 heifers synchronized with the 5 d Co-Synch+CIDR protocol were assigned randomly to receive two injections of FM (FM group; n=158) at 15.5 and 16 d after TAI, or to remain as untreated controls (n=165). Pregnancy per TAI in Experiment 2 was 59.4 and 59.5% at 45 d for control and FM groups, respectively, with no differences between groups (P=0.83). The 5 d Co-Synch+CIDR protocol resulted in an acceptable P/TAI in dairy heifers. However, FM did not improve P/TAI in dairy heifers.

Factors affecting the incidence of postpartum oestrus, ovarian activity and reproductive performance in Thoroughbred mares bred at foal heat under Indian subtropical conditions - Corrected Proof

Sumeet Sharma, M.C.G. Davies Morel, G.S. Dhaliwal — 2010-03-08

Abstract: Decreased reproductive performance due to summer stress is a well known phenomenon in farm livestock. Whether this occurs in the mare and specifically how this might affect postpartum reproductive activity and performance, especially at Foal Heat (FH), is unknown. This study, therefore, aims to investigate this and the factors that might affect postpartum reproductive activity. Reproductive records of 228 Thoroughbred mares (694 mare years) bred in subtropical north-western India were retrospectively analysed. Overt oestrous activity occurred within 21 d postpartum in 92.94% (645/694) of mares. Significantly (p<0.001) more April foaling mares (97.37%, 185/190) expressed postpartum oestrous activity than those foaling in January (83.61%; 51/61) and February (88.49; 123/139). Similarly significantly (p<0.01) fewer multiparous mares failed to demonstrate oestrous activity than primiparous mares (6.12% vs.15.07%; 38/621 vs. 11/73, respectively). 190 of these 694 mares were additionally monitored to confirm ovulation; in these mares onset of FH (oestrus plus confirmed ovulation) occurred 8.42±0.17 d and first ovulation 13.64±0.20 d postpartum. Month, stud farm, year, and parity did not affect interval from parturition to FH onset or to first ovulation; or FH onset to ovulation. In FH bred mares Day 16 pregnancy rate and overall foaling rate were 53.76% (100/186) and 46.24% (86/186) respectively and were similar to those of mares bred later postpartum. FH pregnancy rates were not affected by stud, season, month, year, number of matings, or day of ovulation but were significantly (p<0.008) lowered by increasing mare age. Significantly (p<0.01) lower Day 16 pregnancy rates were observed in uterine treated mares compared to untreated mares (31.09% vs. 57.96%; 9/29 vs. 91/157, respectively), this difference was not evident during the rest of pregnancy. In conclusion, postpartum reproductive and ovarian activity appears to be affected by environment, i.e., delayed in subtropical kept Thoroughbred mares compared to those kept in temperate climates. However, resulting reproductive performance at FH and the factors affecting postpartum reproductive activity are similar.

Sexual behavior of castrated boars treated with prostaglandin F2α - Corrected Proof

2010-03-08

Abstract: The objectives were to test the hypothesis that exogenous prostaglandin F2α (PGF2α) temporarily restores sexual behavior of castrated boars, and to evaluate effects of PGF2α on serum hormone concentrations. At 35 d after castration, nine lean-type adult boars were randomly assigned to three treatments in a 3×3 latin square (with three replicates). Treatments were three doses of PGF2α doses (0, 10, and 20mg) and three periods of treatment, with 5 d between each period. Serum testosterone (T) concentrations were non-detectable at the start of the experiment. Serum concentrations of estradiol (E2), LH, prolactin (PRL), and cortisol were unaffected (P>0.05) by PGF2α treatment. The interval from treatment to ejaculation in boars treated with 10mg (758s) or 20mg (660s) PGF2α did not differ, but were different (P < 0.05) from control boars (>1 800s). Ejaculation duration and false mounts differed (P < 0.05) between control boars and boars treated with 10 or 20mg PGF2α. In conclusion, PGF2α treatment did not change serum concentrations of T, E2, LH, PRL, or cortisol, but restored sexual behavior. This restoration may have been due to an effect of PGF2α directly in specific areas of the brain, or indirectly via release of other hormones that stimulated areas in the brain that affected sexual behavior.

Aneuploidy in rabbit males: semen traits and fertility - Corrected Proof

R. Lavara, M. Baselga, J.S. Vicente — 2010-03-08

Abstract: Chromosomal analyses were performed from peripheral blood samples from 20 adult rabbit males from Line R. This line has been selected for growth rate after weaning through 25 generations. Seminal characteristics and reproductive outcomes from these males were also examined. The chromosomal analysis results showed that one male had an aneuploidy rate of 16% (male A). The aneuploidies found represented both hypo- and hyper- haploidy. Differences between the male A and the contemporary males (males N), in fertility at 12 days post-insemination (44% vs. 66%) and at birth (31% vs. 59%) were observed. The male A also showed a high percentage of pregnancy losses (29% vs. 12%). For seminal characteristics, the percentage of abnormal spermatozoa was statistically different (P<0.05) between male A and males N (34±4 vs. 18±2), and concentration differed significantly between males, showing the male A reduced fewer spermatozoa than males N (67±23 vs. 172±10, x106/ml). Motility and kinematic parameters revealed no differences between males. Differences between male reproductive performance found in this study could be explained by the effect of aneuploidy on spermatogenesis and its deleterious effect on male reproduction.

Influence of different centrifugation protocols on equine semen preservation - Corrected Proof

2010-03-08

Abstract: Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600×g for 10min=CP1) was compared to four protocols with increasing g-force and decreased time period (600×g, 1200×g, 1800×g and 2400×g for 5min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800×g and 2400×g for 5min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P<0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P<0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P<0.005), whereas CP3 and CP5 yielded a lower BCF (P<0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800×g or 2400×g for a shorter period of time (5min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.

Risk factors for clinical endometritis in postpartum dairy cattle - Corrected Proof

2010-03-08

Abstract: Bacterial contamination of the uterine lumen after parturition occurs in most dairy cattle. The presence of clinical endometritis beyond three weeks post partum depends on the balance between microbes, host immunity, and other environmental or animal factors. The present study tested the hypothesis that clinical endometritis is associated with animal factors, such as retained fetal membranes, assisted calving and twins, as well as fecal contamination of the environment. The association between selected risk factors and the lactational incidence risk of clinical endometritis was examined in 293 animals from four dairy herds. Multivariate analysis was used to identify risk factors and quantify their relative risk (RR) and population attributable fraction (PAF) based on the proportion of cows exposed to each factor. The lactational incidence of clinical endometritis was 27% and significant risk factors for clinical endometritis were retained fetal membranes (RR=3.6), assisted calving (RR=1.7), stillbirth (RR=3.1), vulval angle (RR=1.3), primparity (RR=1.8), and male offspring (RR=1.5) but not the cleanliness of the environment or the animal. The highest PAF was associated with male offspring (0.6) so the use of sexed semen has the greatest potential to reduce the incidence of clinical endometritis. The dominant association between retained fetal membranes and clinical endometritis was supported by an expert panel of clinicians. The risk factors for clinical endometritis appear to be associated with trauma of the female genital tract and disruption of the physical barriers to infection rather than fecal contamination.

Differentiation diversity of mouse parthenogenetic embryonic stem cells in chimeric mice - Corrected Proof

2010-03-08

Abstract: Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals.

Short-term storage of canine preantral ovarian follicles using a powdered coconut water (ACP®)-based medium - Corrected Proof

2010-03-08

Abstract: The objective was to investigate the use of powdered coconut water (ACP®)-based medium for short-term preservation of canine preantral follicles. Pairs of ovaries from mongrel bitches (n=9) were divided into fragments. One ovarian fragment, treated as a fresh control, was immediately fixed for histological analysis, whereas the other six ovarian fragments were stored either in phosphate-buffered saline (PBS; control group) or ACP medium in isothermal Styrofoam boxes containing biological ice packs. The boxes were sealed and opened only after 12, 24, or 36h. After opening each box, the ovarian fragments were submitted to histological analysis. In total, 12,302 preantral follicles were evaluated, with 64.5% primordial, 33.3% primary, and 2.3% secondary follicles. There were multiple oocytes in 1.3% of the follicles analyzed. At 24h, ACP was more efficient in preserving follicular morphology than PBS (P<0.05). Compared with the fresh control group, a significant reduction in the percentage of morphologically normal ovarian follicles was observed for PBS, starting at 24h; however, the decline started only at 36h for the ACP medium. During the experiment, the temperature inside the isothermal boxes increased from 3 to 9°C (P<0.05), despite a constant room temperature. In conclusion, powdered coconut water (ACP) was an appropriate medium for short-term storage of canine preantral ovarian follicles.

Glucose uptake and lactate production by the autotransplanted ovary of the ewe during the luteal and follicular phases of the oestrous cycle - Corrected Proof

R.J. Scaramuzzi, B.K. Campbell, C.J.H. Souza, D.T. Baird — 2010-03-02

Abstract: Two experiments were carried out on ewes with ovarian autotransplants to estimate the ovarian uptake of glucose and production of lactate. The first was carried out in the luteal phase of the oestrous cycle. Samples of carotid arterial, ovarian venous and jugular venous blood were collected simultaneously for glucose analysis. The arterial concentration of glucose (58.0 ± 5.0mg/dL; Mean±SEM) was significantly higher than the ovarian venous concentration (42.3±2.4mg/dL; P<0.001). Next, a second more complete experiment was carried out in the luteal and follicular phases of the oestrous cycle. The oestrous cycle was synchronised and samples of carotid arterial, ovarian venous and jugular venous blood were collected simultaneously for glucose and lactate analysis. There were significant positive arterio-venous differences in the concentration of glucose in the luteal (5.6±1.2mg/dL, mean±SEM; P=0.001), early (3.1±0.82mg/d; P=0.003) and late follicular (6.4±1.3mg/dL; P=0.001) phases of the oestrous cycle. There was a significant negative arterio-ovarian venous difference in the concentration of lactate in only the luteal phase (-2.2±0.96mg/dL; P=0.043).The results show significant removal of glucose from the arterial circulation during its passage through the ovary in the luteal, early follicular and late follicular phases of the oestrous cycle. Furthermore, there was lactate production in the luteal phase but not in the follicular phase suggesting that in the luteal phase of the oestrous cycle, ovarian metabolism can be anaerobic.

Comparison of ovulation, fertilization and embryonic survival in low-fertility beef cows compared to fertile females - Corrected Proof

A.C. Warnick, P.J. Hansen — 2010-03-02

Abstract: The objective was to determine physiological causes of low fertility in beef cows. Fertility was compared between low-fertility cows (34 British cows and 64 Brahman crossbred cows; cows that did not get pregnant when mated to fertile bulls in one or two previous breeding seasons); fertile cows (16 Brahman crossbreds; cows having a calf in several of the preceding breeding seasons), and virgin heifers (45 Brahman crossbreds, 2 yr of age). Females were mated to fertile bulls and killed 3 or 34 d after breeding to obtain reproductive tracts. There were no significant differences among groups in rates of ovulation or fertilization. Overall, 14% of females failed to ovulate and 24% that ovulated failed to undergo fertilization. The proportion of cows that were not detected in estrus before Day 34 of pregnancy was lower (P<0.01) for low-fertility British cows (5 of 16 cows, 31%) than for other groups, including low-fertility Brahman crossbred cows (23/32, 72%), fertile cows (8/9, 89%), and heifers (21/24, 88%). All cows that did not return to estrus by Day 34 had an identifiable conceptus. The proportion of conceptuses recovered at Day 34 that were classified as normal (weight and length) was lower (P<0.05) for cows with low fertility (British: 2/5, 40%; Brahman crossbred: 9/23, 39%) than for fertile cows (8/8, 100%) or heifers (18/21; 86%). Similarly, the proportion of cows in which a normal embryo was recovered (cows with normal embryos/number of cows mated) was lower (P<0.001) for low-fertility British cows (2/16, 13%) and low-fertility Brahman crossbred cows (9/32, 28%) than for fertile cows (8/9, 89%) and heifers (18/24, 75%). In conclusion, cows that were infertile in previous breeding seasons did not experience reduced ovulation or fertilization rates, but had greater embryonic mortality. These data highlighted the importance of ovulation and fertilization failure and embryonic mortality as important determinants of pregnancy success. Moreover, increased embryonic loss after Day 34 contributed to infertility in low-fertility cows.

Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors - Corrected Proof

2010-03-02

Abstract: The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 105. The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: A non-random event - Corrected Proof

2010-03-02

Abstract: Although seminiferous tubule maturation in horses begins in the central area of the testis, this process is thought to occur randomly throughout the testis in most mammals. Studies in our laboratory revealed that the establishment of spermatogenesis may not be a synchronous event in the testicular parenchyma of pigs. The objectives of the present study were to evaluate the pattern of seminiferous cord/tubule maturation and the morphological and functional characteristics of testicular somatic cells during postnatal development in three regions of the pig testis: a) near the tunica albuginea (TA); b) in the transitional area between the seminiferous tubules and mediastinum (TR); and c) in the intermediate area (ID) between the TA and TR. Based on the diameter of seminiferous cords/tubules, nucleus size of Sertoli cells and fluid secretion, mainly at 90 and 120 d of age, seminiferous tubule maturation was more advanced in the ID and TR. The mitotic activity of Sertoli cells was higher (P<0.05) in the TR than the ID and TA at 7 and 120 d. Except for the mitotic index of the Leydig cells, which was lower (P<0.05) in the ID at 7, 30, and 180 d than in the TA and TR, other Leydig cell ebd points, e.g., individual cell size, nuclear volume, and cytoplasmic volume, were consistently higher (P<0.05) in the ID, suggesting that steroidogenesis was more active in this region during the period investigated. Overall, we inferred that Leydig cells in the ID may play a pivotal role in postnatal testis development in pigs and this type of cell is likely related to asynchronous testicular parenchyma development, with the transitional area providing the primary zone for growth of seminiferous tubules.

Fetal movements during late gestation in the pig: A longitudinal ultrasonographic study - Corrected Proof

2010-03-02

Abstract: Repeated ultrasonographic observation of fetal movements was used to distinguish movement patterns and to investigate the rate of occurrence and temporal organisation of these patterns (rest-activity cycles) during the last three weeks of gestation in the pig.By means of transabdominal ultrasonography with a 3.5MHz linear array transducer, motility in ten different fetuses (one per sow) was studied. Six (median; range 4–6) 1h recordings were made per fetus at 3–5 day intervals. Fifty-five 1h recordings were available for analysis. The occurrence of fetal general movements (GM), isolated head (HM), forelimb movements (LM), and rotations (ROT) was analysed from video tapes. For each movement pattern, the trend in occurrence over time was assessed by multilevel analysis. The temporal association between different movement patterns was studied by calculation of the kappa value.ROT occurred very infrequently and showed no particular trend over time. GM, HM, and LM showed a significant decreasing trend towards parturition (P<0.01). Total fetal activity (i.e., the sum of the four movement incidences) declined from an average of 25% of recording time to 9% over the last three weeks of pregnancy. Periods of fetal quiescence gradually increased with progressing gestation (P<0.05). There was no evidence of concordant association between the periods of rest and activity of GM, HM, and LM or of improved temporal linkage between these movement patterns with time.Fetal bodily activity decreases towards parturition mainly due to prolonged periods of rest. Fetal movement patterns show rest-activity cycles, but each pattern appears to cycle independently from the other throughout late gestation. The present results of spontaneous fetal movements in the pig provide reference data for future studies of fetal activity under different zootechnical conditions or pharmacological interventions.

Differential gene expression in bovine elongated (Day 17) embryos produced by somatic cell nucleus transfer and in vitro fertilization - Corrected Proof

2010-03-02

Abstract: Somatic cloning in cattle is associated with impaired embryo development, caused by inappropriate epigenetic reprogramming during embryogenesis; however, there is a paucity of data regarding gene expression at the critical elongation and peri-implantation stages. The objective of the present study was to identify genes differentially expressed in bovine cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or IVF). Day 7 blastocysts (Hand Made Cloned or IVP) were transferred to recipient cattle and collected at Day 17. The efficiency of recovery of elongated embryos was similar, however cloned embryos elongated less than IVP embryos (91.8±45.8 vs. 174±50mm) and fewer had embryonic discs (63 vs. 83%). Qualitative and quantitative PCR detected expression of OCT4, NANOG, IFNtau, EOMES, FGF4, SOX2, and CDX2 in all IVP embryos. In most cloned embryos, NANOG and FGF4 were absent (verified by qPCR); NANOG, EOMES, and FGF4 were underexpressed, whereas IFNtau was overexpressed in cloned embryos. Based on qPCRs, other genes, i.e., SPARC, SNRB1, and CBPP22, were down-regulated in cloned embryos, whereas HSP70 and TDKP1 were overexpressed. In bovine microarrays, 47 genes (3.6%) were deregulated in cloned embryos, including several involved in trophoblast growth and differentiation. In conclusion, we inferred that these data were indicative of incomplete epigenetic reprogramming after cloning; this could lead to aberrant gene expression and subsequently early pregnancy loss. There was an apparent association between incomplete morphological elongation and aberrant reprogramming of a subset of genes critical for early embryonic development.

Seasonal infertility in sows: A five year field study to analyze the relative roles of heat stress and photoperiod - Corrected Proof

2010-03-02

Abstract: The objective of this study was to analyze the relative roles of high temperature and photoperiod as environmental factors of seasonal infertility in swine. The results of five years (2003–2007) of ultrasound pregnancy diagnosis carried out in 266 indoor farms were analyzed. For all farms, the data covered the entire study period. The farms were situated in four French regions. The data of 22,773 batches and 610,117 sows were included. Seasonal infertility was defined as the relative difference between the fertility rate in ‘summer’ (inseminations in weeks 25–42) and ‘winter’ (inseminations in weeks 1–18 of the same year). In each region, two meteorological variables were defined, based on the data of a reference weather station: the number of hot days (maximum temperature ≥ 25°C) and tropical days (maximum temperature ≥ 32°C and minimum temperature ≥ 18°C). The mean fertility was 85%. The median seasonal infertility was 2.8% and more than 7.1% for a quarter of farms. Seasonal infertility did not vary with areas or baseline fertility (defined for each studied farm as the average winter fertility over five years). Seasonal infertility differed with the year (p<0.001). Seasonal infertility was significantly higher during 2003 than in the other four years, which did not differ among each other. In the four regions, 2003 was the year with the highest number of hot days and 2007 with the least. Our study strengthens the hypothesis of a prominent role of photoperiod in seasonal infertility and of an additional role of heat stress the hottest years.

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm - Corrected Proof

M. Suquet, C. Labbe, R. Brizard, A. Donval, J.R. Le Coz, C. Quere, P. Haffray — 2010-03-02

Abstract: Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation.A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.

Seasonality of reproduction in wild boar (Sus scrofa) assessed by fecal and plasmatic steroids - Corrected Proof

2010-02-24

Abstract: The collection of biological samples through non-invasive techniques represents one way of monitoring in vivo physiological changes associated with reproductive activity. Such techniques are particularly important for the study of animal species in the wild.The goals of this study were 1) to evaluate fecal progestogen (P), estrogen (E), and androgen (A) by means of radioimmunoassays, in male and female wild boars culled in the Piedmont, Italy area; 2) to compare them with plasmatic concentrations and the animals’ reproductive status; and 3) to assess variations in reproductive seasonality between two populations of wild boars living in a mountainous vs. a plain habitat in Piedmont.The results demonstrate a positive correlation between fecal and plasmatic steroid concentrations (r=0.46, 0.58, and 0.45 for plasma P4 and P, E2 and E, and T and A; P<0.05). Moreover, high fecal levels of both P and E (>170ng/g and >100pg/g respectively) were found in 70.6% of pregnant sows and in none of the non-pregnant animals, thus supporting the use of this technique for detecting pregnancy status in wild boar.Similar birth patterns were displayed by the mountain and plain populations, but births peaked significantly only in the mountain population, in the spring (46%, P<0.05, vs. other seasons). A corresponding autumnal peak of plasma testosterone concentrations in males was displayed only by the mountain population (7.4 vs.<2.0ng/mL in the other seasons, P<0.05).The correlation between fecal and plasmatic steroid concentrations obtained in this study supports the applicability of this non-invasive sampling technique for monitoring reproductive status in wild boar, thus enabling a more informed and correct management of the species.

Comparison of three commercial vaccines for preventing persistent infection with bovine viral diarrhea virus - Corrected Proof

2010-02-24

Abstract: Eighty crossbred beef heifers were randomly allocated to four groups to evaluate the efficacy of vaccination in preventing development of calves persistently infected with bovine viral diarrhea virus (BVDV). Group 1 (n=11) was non-vaccinated controls, whereas three groups were vaccinated with commercially available multivalent BVDV vaccines at weaning (∼7 mo of age), 28 d post-weaning, ∼1 y of age, and 28 d later. Groups 2 (n=23) and 3 (n=23) were given a modified-live BVDV vaccine, whereas Group 4 was given an inactivated BVDV vaccine. Heifers were bred by AI and subsequently exposed to two bulls. At 61 d after AI, 70 heifers were pregnant (n=10 for Group 1 and n=20/group for Groups 2, 3, and 4). Three cattle persistently infected with BVDV were commingled with the pregnant heifers (in an isolated pasture) from 68 to 126 d after AI. Thereafter, viremias were detected in pregnant heifers from Groups 1, 3, and 4 (10/10, 1/20, and 10/20, respectively), but not in pregnant heifers from Group 2 (0/20). Resulting calves were assessed for persistent infection using serum PCR, ear notch antigen capture-ELISA, and immunohistochemistry. Persistently infected calves were only produced in Group 1 (10/10) and Group 4 (2/18). In conclusion, commercial vaccines provided effective fetal protection despite prolonged natural exposure to BVDV. Given that viremias were detected in 11 vaccinated heifers after BVDV exposure, and two vaccinated heifers gave birth to persistently infected calves, there is continued need for biosecurity and diagnostic surveillance, in addition to vaccination, to ensure effective BVDV control.

Generation of a recloned transgenic cat expressing red fluorescence protein - Corrected Proof

S.J. Cho, J.I. Bang, X.F. Yu, Y.S. Lee, J.H. Kim, J.T. Jeon, S.T. Yee, I.K. Kong — 2010-02-22

Abstract: Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21±7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications.

Hormonal concentrations in bitches with primary uterine inertia - Corrected Proof

A. Bergström, B. Fransson, A.-S. Lagerstedt, H. Kindahl, Ulf Olsson, K. Olsson — 2010-02-22

Abstract: Normal labor is accompanied by sequential changes in blood concentrations of prostaglandin F2α (measured as 15-ketodihydro-PGF2α=PGFM), progesterone, estradiol, oxytocin, vasopressin, and of elevated cortisol levels. The aim of this study was to investigate hormone concentrations in dogs diagnosed with primary uterine inertia before and during treatment by cesarian section. The hypothesis was the dogs would have abnormally low plasma concentrations in one or several of the hormones involved in parturition. The study comprised seven bitches with total primary uterine inertia (dystocia group) treated with cesarian section and six healthy bitches (control group) subjected to planned cesarean section. Blood samples were taken before anesthesia, before surgery started, on delivery of the first puppy and on delivery of the last puppy. The progesterone:PGFM ratio in plasma was higher in the dystocia group than in the control group, but the serum estradiol concentration did not differ between groups. The plasma concentrations of oxytocin and vasopressin increased in both groups when the first puppies were delivered, but both hormones were more elevated in the control group than in the dystocia group on delivery of the last puppies. The plasma cortisol concentration increased to the same level in both groups. In conclusion, the ratio between progesterone and PGFM was higher and the oxytocin and vasopressin concentrations lower in the dystocia dogs than in the control dogs. The findings indicate that these hormones are involved in the pathophysiology of total primary uterine inertia in bitches.

Substantiation of Ovarian Effects of Leptin by Challenging a Mouse Model of Obesity/ Type 2 Diabetes - Corrected Proof

2010-02-22

Abstract: The goal of the current was to elucidate if treatment with gonadotrophins and leptin can circumvent infertility in obese mice and to establish whether reproductive effects of leptin are influenced at the hypothalamus-hypophysis or ovarian level by using a leptin deficient mouse model of obesity/type 2 diabetes (ob/ob) treated with leptin. The ovulatory response and the fertilization success were compared with the results obtained in ob/ob dams pretreated with a gonadotrophin-replacement therapy or in two groups (ob/ob and wild-type) of control non-pretreated females. The number of corpora lutea was significantly lower in control ob/ob mice than in wild-type dams. Treatment with gonadotrophin-replacement therapy did not increase significantly the ovulation rate in ob/ob, but the administration of leptin-replacement treatment allowed the authors to obtain a number of corpora lutea and oocytes/zygotes similar to those obtained in wild-type females. Furthermore, the leptin supply succeeded in producing fertilized zygotes, although in a lower number than found in the wild-type control. Thus, the hypogonadotrophic state in obese mice may be circumvented by the administration of a gonadotrophin-replacement therapy combined with a protocol for controlled ovarian stimulation, but fertile ovulations are only obtained after applying leptin-replacement therapy. Current results strongly support the existence of direct local effects of leptin on the ovary.

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B - Corrected Proof

L. Zhang, Y. Liang, Y. Liu, C.-L. Xiong — 2010-02-22

Abstract: Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2h (P=0.0006) and 3h (P<0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16h of in vitro maturation (P=0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat - Corrected Proof

2010-02-22

Abstract: Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330μg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n=17) or a placebo (PLC; n=7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean±SEM) 7.0±1.3 and 7.0±1.7 d after treatment (P>0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P<0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P<0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4±1.7 and 120±17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n=3), mid pregnancy (from 26 to 45 d; n=4), late pregnancy (> 45 d; n=3), or injection of a placebo in early (n=1), mid (n=2), or late pregnancy (n=1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P4) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P4 concentrations did not differ among treatment groups (P>0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis) - Corrected Proof

M.A. Stoops, M.W. Atkinson, E.S. Blumer, M.K. Campbell, T.L. Roth — 2010-02-22

Abstract: The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n=6; 7.5–34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75–100 mAmps; 4–10 volts; 7–150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516×106/mL) and motility (79%). Post-thaw sperm characteristics were higher (P<0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50–55% compared to 22–37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification - Corrected Proof

2010-02-22

Abstract: Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24h of culture) was improved (P<0.05) by the addition of 10μM Y-27632 (94.9±2.4%, mean±SEM) compared to a control (78.0±6.0%). Conversely, after 48h of culture, there were no significant differences in hatching rate (62.8±11.1 vs. 59.6±9.4%) and mean total cell number (135.2±13.1 vs. 146.7±13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7±3.8 vs. 54.7±8.9%, P<0.05), with no difference in mean total cell number of blastocysts (230.0±23.0 vs. 191.2±22.2, P=0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5±2.1, 31.4±2.3, 36.2±3.2, and 28.6±6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods - Corrected Proof

2010-02-22

Abstract: Stallion sperm from semen collected in Southern Italy during the breeding (June–July) and non-breeding (December–January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc, with Galβ1,3GalNAc, α/βGalNAc and glycans with terminal/internal αMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galβ1,4GlcNAc (Ricinus communis120 affinity) (RCA120) and L-Fucα1,2Galβ1,4GlcNAcβ (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and αL-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with αGalNAc, GlcNAc, and L-Fucα1,2Galβ1,4GlcNAcβ (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

Environmental influences on the production of pre-implantation embryos - Corrected Proof

2010-02-22

Abstract: Generation and cryopreservation of transgenic mice depend on reliable and continuous production of pre-implantation embryos. To suppress circannual and circadian rhythms driving the physiological and sexual behaviour of free living animals, laboratory animals are housed under standardized conditions. It remains to be elucidated if the artificial climate can cover all environmental effects. Here, we report that the humidity in an animal facility affects the embryo yield. The weather at the location of the facility, especially the temperature, influences the climate within an animal facility; weather peaks are obviously covered in part only, even if the facility is equipped with a powerful air-conditioning supply. Subsequently, external weather changes interact with the environment within the facility, influencing the production of embryos. Furthermore, noise and/or vibrations as generated by construction works, negatively affect the embryo yield.

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs - Corrected Proof

A. Blitek, A. Waclawik, M.M. Kaczmarek, J. Kiewisz, A.J. Ziecik — 2010-02-22

Abstract: Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE2, PGF2α, and PGFM (PGF2α metabolite) content, as well as PGE2 synthase (mPGES-1) and PGF2α synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10–11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE2 in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE2 content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE2 accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF2α content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.

Intra-cervical application of Misoprostol at estrus alters the content of cervical hyaluronan and the mRNA expression of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and cyclooxygenase-2 in the ewe - Corrected Proof

S. Leethongdee, C.M. Kershaw-Young, R.J. Scaramuzzi, M. Khalid — 2010-02-22

Abstract: The complex anatomy the of ovine cervix limits the success of transcervical artificial insemination in sheep, but Misoprostol (a PGE1 analogue) relaxes the cervix and facilitates transcervical artificial insemination. However, the mechanism by which Misoprostol causes cervical relaxation is not known. This study examined if intra-cervical Misoprostol altered the hyaluronan content and the mRNA expression of COX-2, LHR, or FSHR in the cervix of the estrus ewe. Estrus was synchronized in cyclic ewes with progestagen pessaries and 48h after sponge removal ewes were treated intra-cervically with 0 (controls), 200, or 400μg Misoprostol. Hyaluronan content was determined by ELISA and mRNA expression of LHR, FSHR, and COX-2 was analyzed by in situ hybridization using digoxigenin-11-uridine-5′-triphosphate labeled riboprobes. The hyaluronan content of the cervix was significantly higher in sheep that received 200 (P<0.05) or 400 (P<0.05) μg Misoprostol compared to controls. Moreover, it was significantly (P<0.05) higher in the vaginal region compared to mid and uterine regions. Misoprostol increased (P<0.05) the mRNA expression of LHR and COX-2 but not FSHR. The expression for all three genes was highest in the vaginal region and lowest in uterine region. The luminal epithelium and circular smooth muscle layers had higher (P<0.05) expression for LHR, FSHR, and COX-2 mRNAs, and the sub-epithelial stroma had the lowest (P<0.05). We propose that the intra-cervical application of Misoprostol induces the mRNA expression of LHR, FSHR, and COX-2 through a positive feedback loop. The data suggest that softening of the cervix by Misoprostol is caused by an increase in the hyaluronan content of the cervix.

Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach - Corrected Proof

A.M. Petrunkina, D. Waberski, H. Bollwein, H. Sieme — 2010-02-22

Abstract: Flow cytometry is now being used more frequently to determine sperm functional characteristics during semen assessment for artificial insemination. With this methodology, viable and potentially functional cells are detected as unstained events differentiated from non-sperm events through their light-scattering characteristics. However, it can be shown mathematically that identification of sperm on the basis of light scatter leads to significant overestimation of unstained viable cells and underestimation of responding cells in tests of sperm function (subpopulations expressing different fluorescence patterns). We have developed a simple and cost-efficient flow cytometric approach for identifying non-sperm particles that can be carried out in parallel with functional assessments. Our method is based on the sperm's osmotic intolerance. Diluted in water, lethal osmotic shock causes major damage to the cell membranes, and all sperm will stain with propidium iodide (PI). Particulate material which is not PI-positive can then be quantitatively evaluated by FACS analysis and the results substituted in mathematical equations to provide true values for sperm counts and subpopulations. In practical tests, the percentage of non-sperm particles determined by this technique was closely comparable to the figure obtained either by SYBR14®/PI staining or by PI/CFDA staining. As well as being valuable with respect to tests of sperm function, the procedure is also suitable for obtaining accurate sperm counts during routine semen evaluation.

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity - Corrected Proof

Z. Nur, B. Zik, B. Ustuner, H. Sagirkaya, C.G. Ozguden — 2010-02-22

Abstract: This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5mM sucrose or 62.5mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5°C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5°C, dilution, equilibration, and thawing) had negative effects on motility (P<0.001), acrosome integrity (P<0.001), and DNA integrity as determined by AO (P<0.001) and TUNEL (P<0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P<0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.

Calcium, parathyroid hormone, oxytocin and pH profiles in the whelping bitch - Corrected Proof

2010-02-22

Abstract: Despite the high prevalence of primary uterine inertia in whelping bitches, the underlying pathogenesis remains unclear. The objectives were to i) determine serum concentrations of total calcium, ionized calcium (iCa), parathyroid hormone (PTH), and blood pH in normally whelping bitches throughout the peri-parturient period; and ii) investigate relationships among iCa, PTH, and acid-base status, and the role that they and oxytocin may have in the underlying pathogenesis of canine uterine inertia. Bitches were randomly selected from a population of German Shepherd Dog bitches with a history of uncomplicated parturition (Group 1; n=10), and from a population of Labrador bitches with a clinical history of an increased incidence of uterine inertia and stillbirths (Group 2; n=20). Jugular blood samples were collected daily from -4 d to the onset of whelping (t=0h), and then every 4h until the last pup was born. Overall, bitches from Group 2 had higher mean±SEM serum concentrations of PTH (4.72±2.45pmol/L, P<0.001), lower iCa (1.31±0.08pmol/L, P<0.05), and higher venous pH (7.41±0.03, P<0.005) than bitches from Group 1 (2.9±1.44pmol/L, 1.38±0.06mmol/L, and 7.33±0.02, respectively) during the periparturient period. However, there was no significant difference between Groups 1 and 2 for serum oxytocin concentrations during the periparturient period (45.5±40 and 65.5±82pg/mL). We inferred that low iCa resulting from a rising pH and decreasing PTH during the periparturient period may have contributed to decreased uterine contractility and increased risk of stillbirths. Therefore, manipulating the cationic/anionic difference in diets of pregnant bitches, similar to the bovine model for hypocalcamia, may reduce the incidence of stillbirths in the bitch.

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm - Corrected Proof

K. Chatdarong, P. Thuwanut, J.M. Morrell — 2010-02-22

Abstract: The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, ∼200×106 sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300×g for 20min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean±SD of 16.4±8.7, 10.7±8.9, and 2.3±1.7%, respectively; P<0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02±0.01, 0.02±0.04, 0.03±0.04, and 0.44±0.22×106cells/mL, respectively; P<0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P>0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P<0.05), due to the lowest proportion of coiled tails (P<0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P>0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.

Extremely low frequency electromagnetic field exposure affects fertilization outcome in swine animal model - Corrected Proof

2010-02-22

Abstract: Modern society continuously exposes the population to electromagnetic radiation, the effects of which on human health, in particular reproduction, are still unknown. The aim of this research was to assess the effect of acute (1h) exposure of boar spermatozoa to a 50Hz extremely low frequency electromagnetic field (ELF-EMF) on early fertility outcome. The effect of intensities ranging from 0 to 2mT on morpho-functional integrity of capacitated spermatozoa was examined in vitro. The oviducts containing or without spermatozoa were then exposed to the minimum in vivo, TD50, and maximum intensities determined in vitro, 4h before ovulation. The effects of ELF-EMF on spermatozoa in terms of early embryo development were evaluated after 12h and 6 days. It was found that in vitro ELF-EMF >0.5mT induced a progressive acrosome damage, thus compromising the ability of spermatozoa to undergo acrosomal reaction after zona pellucida stimulation and reducing the in vitro fertilization outcome. These effects became evident at 0.75mT and reached the plateau at 1mT. Under in vivo conditions, the ELF-EMF intensity of 1mT was able to compromise sperm function, significantly reducing the fertilization rate. In addition, the exposure of oviducts to fields ≥ 0.75mT in the absence of spermatozoa was able to negatively affect early embryo development. In fact, it was found to cause a slowdown in the embryo cleavage. In conclusion, it was demonstrated how and at which intensities ELF-EMF negatively affect early fertility outcome in a highly predictive animal model.

Evaluating the effectiveness of different treatments of uterine infections in female camels (Camelus dromedarius) - Corrected Proof

A. Ali, F.A. Al-sobayil, A. Al-Hawas — 2010-02-22

Abstract: A total of 480 female camels with a history of conception failure were examined through transrectal palpation, ultrasonography, and vaginal exploration. Animals were categorized according to parity (nulliparous n=200 vs. multiparous n=280), and type of uterine infection (endometritis n=360 vs. metritis n=120). They were randomly assigned to receive one of three intrauterine treatments: (i) 100mL acriflavin 0.1% (group 1, n=170), (ii) 100mL lotagen 4% (group 2, n=200), or (iii) 300mg/100mL gentamicin sulphate (group 3, n=110). All groups received 500μg cloprostenol IM at infusion. Animals were exposed for breeding 7 d later and received 5000 IU hCG im at mating. The criteria for efficacy of treatment were 90 days non-return rate (90 d NRR) and calving rate (CR). The results showed that the 90 d NRR and CR were significantly influenced by parity, type of uterine infection, regime of treatment, and their interactions, P<0.05. Treatment regimes were approximately equally efficient in treating females with endometritis (90 d NRR were 64%, 53.1% and 53.3% and CR were 58.9%, 49.3%, and 42.5% for groups 1, 2, and 3, respectively, P>0.05). In contrast, regimes differed in treating those with metritis (90 d NRR were 55.6%, 75%, and 28.6% and CR were 31.6%, 54.8%, and 12.5% for groups 1, 2, and 3, respectively, P<0.05). In conclusion, a regime consisted of intrauterine lotagen infusion and administration of PGF2α at infusion and hCG at mating was more efficient for treating female camels with metritis.

Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination - Corrected Proof

2010-02-22

Abstract: Cytogenetic analysis of 58 boars at an artificial insemination (AI) centre revealed the presence of a reciprocal chromosome translocation, rcp(1;11)(q−;p+), in two Duroc boars. Pedigree analysis of these two boars suggested familial transmission of the chromosome rearrangement. The reproductive consequences of this translocation were determined in a herd of sows that had received semen doses from these and other boars. All sows underwent multiple AI, with different groups established retrospectively depending on the percentage of semen doses provided by the carrier boars ([number of carrier boar doses/total number doses provided] x 100): 0%, 25%, 50%, 75%, 100%. The fertility rates (percentage of successful multiple AIs/total multiple AIs) recorded for multiple AI including semen doses from the carrier boars were not significantly different from those recorded when all semen doses were supplied by normal-karyotype boars. A reduction in litter size of 29.38% was observed, however, in litters sired by one of the carrier boars when its participation in multiple AI was 100%. The number of live-born piglets per litter gradually decreased (P<0.05) as the percentage participation in multiple AI (25, 50, or 75%) of the carrier boar increased. In addition, both carrier boars sired some piglets with signs of cleft palate and complex malformations of the front legs; these died soon after birth. In conclusion, the boars carrying the translocation rcp(1;11)(q−;p+) showed reduced reproductive performance.

Method agreement analysis: A review of correct methodology - Corrected Proof

P.F. Watson, A. Petrie — 2010-02-08

Abstract: The correct approach to analyzing method agreement is discussed. Whether we are considering agreement between two measurements on the same samples (repeatability) or two individuals using identical methodology on identical samples (reproducibility) or comparing two methods, appropriate procedures are described, and worked examples are shown. The correct approaches for both categorical and numerical variables are explained. More complex analyses involving a comparison of more than two pairs of data are mentioned and guidance for these analyses given. Simple formulae for calculating the approximate sample size needed for agreement analysis are also given. Examples of good practice from the reproduction literature are cited, and common errors of methodology are indicated.

Developmental competence and mRNA expression of preimplantation in vitro–produced embryos from prepubertal and postpubertal cattle and their relationship with apoptosis after intraovarian administration of IGF-1 - Corrected Proof

2010-02-08

Abstract: Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1–treated preP group (11.2±2.4%), the control preP group (10.4±3.0%), and in the IGF-1 postP group (10.9±2.3%). These were lower (P≤0.01) compared with the control postP group (21.2±3.8%) and with cows (23±3.7%). The expression profile of the six genes was partly affected by age and IGF-1 treatment. Apoptosis was correlated with the age of the oocyte donors and was increased in blastocysts derived from prepubertal heifers. Results show that apoptosis is a critical feature of the acquisition of developmental competence of oocytes from prepubertal cattle and that IGF-1 did not beneficially affect oocyte developmental competence.

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo–derived bovine embryos - Corrected Proof

2010-02-03

Abstract: Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo–derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo–derived bovine embryos (n=10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID50)/mL. Additionally, control heifers received 1.5×106 CCID50 BVDV/.5mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro - Corrected Proof

R. Gualtieri, V. Mollo, V. Barbato, R. Talevi — 2010-02-03

Abstract: In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro–cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: A useful tool for reproductive diagnosis in the bitch - Corrected Proof

D. Groppetti, A. Pecile, S. Arrighi, A. Di Giancamillo, F. Cremonesi — 2010-02-01

Abstract: New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch (Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N=35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N=10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.

Clinical use of human chorionic gonadotropin in dairy cows: An update - Corrected Proof

F. De Rensis, F. López-Gatius, I. García-Ispierto, M. Techakumpu — 2010-02-01

Abstract: Human chorionic gonadotropin (hCG) has a potent luteinizing hormone (LH)-like effect in cattle that extends the life span of the corpus luteum (CL) and increases progesterone synthesis, induces ovulation throughout the estrous cycle, promotes the formation of accessory corpora lutea when applied in the early luteal phase, and modifies follicular wave dynamics increasing the frequency of three-wave dominant follicular cycles. As hCG acts on ovarian cells independently of the pituitary gland and its effect is longer lasting than that produced by endogenous LH release, use of hCG rather than gonadotropin-releasing hormone (GnRH) could be targeted at populations of subfertile cows. This review describes the clinical use of hCG to improve the reproductive performance of dairy cows. In addition, we describe recent developments in the therapeutic use of hCG and studies addressing the benefits of including hCG in estrus and ovulation synchronization protocols. Our review ends with a critical discussion of how earlier findings related to ovarian responses to hCG treatment can be interpreted in the light of recent advances in the clinical applications of hCG.

Extraction forces in bovine obstetrics: An in vitro study investigating alternate and simultaneous traction modes - Corrected Proof

M. Becker, G. Tsousis, M. Lüpke, F. Goblet, C. Heun, H. Seifert, H. Bollwein — 2010-02-01

Abstract: Whether extraction of a calf in longitudinal anterior presentation should be carried out by simultaneous or alternate traction on the forelimbs remains controversial. Because most recommendations are based on empirical observations rather than on scientific studies, the aim of this study was to develop an in vitro model to objectively compare the forces occurring during alternate and simultaneous traction. In a biomechanical in vitro model, 12 dead Holstein-Friesian (Bos taurus) calves were pulled through the prepared pelvic specimen of a cow at a controlled speed using two electric motors. Traction was applied simultaneously (ST) to both legs or alternately (AT) to one leg at a time to advance the calf 5 cm (AT 5) or 10 cm (AT 10). Forces on each limb were measured digitally using load cells. In all cases, two peaks of maximum force occurred during the extraction of the cranial part of the body. The first peak was observed when the elbows were pulled into the pelvis, and the second peak occurred when the chest emerged from the pelvis. Up to and including entry of the elbows into the pelvis, the maximum force on a single limb (341±106 N) was lowest (P<0.01) using AT10. The maximum traction forces acting on a single limb using AT5 (411±86 N) and ST (431±127 N) did not differ (P>0.05). During extraction of the thorax, the maximum force acting on a single limb was lower (P<0.0001) using ST (352±98 N) compared with AT5 (432±79 N) and AT10 (547±115 N). Based on these findings, alternate-limb traction, 10cm at a time, should be used until both elbows have entered the pelvis. Simultaneous traction should then be applied to both forelimbs to complete extraction of the chest.