Theriogenology - Articles in Press

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.

Preface - Corrected Proof

Ann Van Soom, Alireza Fazeli — 2010-02-04

Maternal communication with gametes and embryos stands at the base of early embryonic development, implantation, and maintenance of a pregnancy. This interaction not only underpins pregnancy but also influences the future health of the offspring. Therefore, understanding the mechanisms involved in the communication between the maternal tract and gametes and embryos in terms of the molecules involved and their function is of major scientific, economic, and health importance. Hence, with the support of the European Union, a COST ACTION program (FA0702; www.cost-gemini.eu) entitled “Maternal Interaction with Gametes and Embryos” was launched on 28 February 2008. The main objective of the action is to establish a network of researchers working on different aspects of maternal interaction with gametes and embryo in different species to advance toward creation of a so-called interactome map of cell-to-cell interactions as well as endocrine and paracrine interactions between gametes, embryos, and female reproductive tract during different stages of the reproductive cycle and pregnancy at health and under disturbed maternal nutrition and metabolism. Some of the specific aims of this action are as follows:

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.

Spatial and temporal expression of spermadhesin genes in reproductive tracts of male and female pigs and ejaculated sperm - Corrected Proof

C. Song, B. Gao, H. Wu, X. Wang, G. Chen, J. Mao — 2010-01-27

Abstract: Spermadhesins, a novel protein family identified in the reproductive tract of ungulates, have important roles in reproduction. In this study, the expression of pig (Sus domesticus) spermadhesion genes in seminal vesicles, prostate, and bulbourethral glands from birth to sexual maturity and the spatial expression in adult male and female genital tracts and ejaculated sperm of Meishan pigs were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). In general, all spermadhesin genes increased from Days 1 to 150 in the seminal vesicle and bulbourethral gland. However, their expression in the prostate was variable; it increased from Days 1 to 60 and then declined until Day 150. In adult boars, all genes had a very high level of expression in the seminal vesicle and somewhat lower (but still relatively high) in the prostate, caput and caudal epididymides, and bulbourethral gland. Expression of AQN1 and AQN3 was not detectable in the corpus epididymis. In the testis, AQN3 gene expression was not detectable, and gene expressions were weak for AQN1, PSP-I, and PSP-II, but strong for AWN. In female pigs, most spermadhesins had low expression in the cervix, uterine horn, oviduct, and ovary. Expression of AQN1 and AQN3 was very weak in the cervix and uterine horn. Signals for AQN1 in oviduct and ovary and AQN3 in ovary were not detectable, whereas AWN had high expression in the cervix and uterine horn. In ejaculated sperm, a strong mRNA signal of spermadhesins was detected. We concluded that transcripts of spermadhesins were not only distributed extensively in male and female reproductive tissues but also in ejaculated sperm. Furthermore, their dynamic changes of expression paralleled reproductive development. Seminal vesicles were the main source of spermadhesins; when the boar reached puberty, expression of spermadhesins reached very high levels.

Determination of heart rate and heart rate variability in the equine fetus by fetomaternal electrocardiography - Corrected Proof

C. Nagel, J. Aurich, C. Aurich — 2010-01-27

Abstract: Heart rate is an important parameter of fetal well-being. We have analyzed fetal heart rate (HR) and heart rate variability (HRV) by fetomaternal electrocardiography (ECG) in the horse (Equus caballus) from midpregnancy to foaling. It was the aim of the study to detect changes in the regulation of fetal cardiac activity over time and to establish normal values in undisturbed pregnancies. A total of 22 mares were available for the study. Fetomaternal electrocardiography was a reliable technique to detect cardiac signals in fetuses between Day 173 of gestation and foaling. Fetal HR decreased from 115±4 beats/min (Days 170 to 240 of gestation) to 83±3 beats/min (Day 320) to 79±1 beats/min (1 d before foaling; P<0.001). Mean beat to beat (RR) interval and standard deviation of the RR interval (SDRR) increased (P<0.001). Gestational age thus affects RR interval and HR in the equine fetus. From Days 270 to 340 of gestation, SDRR increased from 11.4±1.3 msec on Day 270 to 27.8±3.6 msec on Day 340 (P<0.05), and the root mean square of successive RR differences (RMSSD) tended to increase (P=0.07), indicating maturation of the fetal autonomous nervous system. For the last 10 d before foaling, fetal HR and HRV remained constant and did not allow predicting the onset of parturition in the horse. Only during the last 30min before the foal was born, in 4 of 5 fetuses, HR decreased and RR interval increased. Accelerations and decelerations in HR were detectable at all times, but neither their number nor duration changed over time.

Open pulled straw vitrification of goat embryos at various stages of development - Corrected Proof

A.N. Al Yacoub, M. Gauly, W. Holtz — 2010-01-27

Abstract: This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.

Epidermal growth factor can be used in lieu of follicle-stimulating hormone for nuclear maturation of porcine oocytes in vitro - Corrected Proof

S.J. Uhm, M.K. Gupta, J.H. Yang, H.-J. Chung, T.S. Min, H.T. Lee — 2010-01-27

Abstract: Epidermal growth factor (EGF) has been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes, but inconsistencies exist between earlier studies, probably due to differences in the culture conditions used. Using a serum- and hormone-free in vitro maturation (IVM) medium, this study investigated the specific contribution of EGF on IVM of porcine (Sus scrofa) oocytes and its interactive effects with follicle-stimulating hormone (FSH), porcine follicular fluid (pFF), cumulus cells, and serum. It was noteworthy that EGF functionally mimicked the action of FSH and could completely replace FSH for nuclear maturation (83.2±4.4% vs. 55.9±5.2%; mean±SEM), whereas EGF had a synergistic effect with FSH on cytoplasmic maturation of porcine oocytes (P<0.05). Specific inhibition of EGF receptor (EGFR) by tyrphostin AG 1478 inhibited both EGF- and FSH-induced meiotic resumption (17.9±5.2% and 18.2±4.4%, respectively), thereby suggesting that EGFR signaling pathway was essential for oocyte reentry into the meiotic cell cycle. Furthermore, it is possible that FSH action occurs via the EGFR signaling pathway to induce meiotic maturation, although alternate pathways could not be excluded. There were also individual or combined effects of cumulus cells, FSH, serum, and pFF with EGF on IVM of porcine oocytes (P<0.05). Although FSH had a synergistic effect with EGF on cytoplasmic maturation, pFF masked the effects of EGF on both nuclear and cytoplasmic maturation of porcine oocytes (P<0.05). Moreover, the presence of cumulus cells was essential for EGF action. In conclusion, a defined system was used to better examine the effects of EGF. We inferred that EGF functionally mimics FSH for nuclear maturation of porcine oocytes, and its exogenous supplementation into IVM medium can optimize the beneficial effects of FSH on cytoplasmic maturation of oocytes to obtain enhanced embryo development in vitro.

Evaluation of the clinical efficacy of Gonazon implants in the treatment of reproductive pathologies, behavioral problems, and suppression of reproductive function in the male dog - Corrected Proof

2010-01-25

Abstract: Efficacy of a slow-release gonadotropin-releasing hormone (GnRH)-agonist implant (Gonazon) was assessed in 53 male dogs presented with benign prostatic hyperplasia (BPH), hypersexuality, aggressive behavior (either alone or in combination), excessive micturition, or to suppress fertility. Changes in testosterone (T) and estradiol (E2) concentrations and size of testes and prostate were monitored on Weeks 0, +8, and +26 after implantation. Additional measurements during and after this period were performed in 35 dogs. Clinical signs were assessed by the owners. All implants except one were retained throughout the study. Full downregulation of testicular function (T<0.35 nmol/L) was achieved in 46 dogs, five dogs showed partial downregulation (T = 0.36 to 0.47 nmol/L), one dog did not respond, and another one displayed a transient downregulation on Week +18. On Week +8, mean T and E2 levels were reduced by 96% and 62%, respectively, and did not further decrease. Full downregulation (T<0.35 nmol/L) lasted between 6 to >22 mo in most dogs except two. Compared with pretreatment values, mean testicular and prostatic size was reduced (P<0.00001) by 54% and 52%, respectively, on Week +8 and by 68% and 64%, respectively, on Week +26. Relative reduction of prostatic size was more marked in dogs with BPH than in healthy ones on Week +8 (P<0.05) and Week +26 (P<0.02), and clinical signs of BPH disappeared rapidly after implantation. Dogs affected with BPH were significantly older (P<0.001) than nonaffected ones (9.7 vs. 2.5 yr). Hypersexuality was more common in dogs<3 yr of age, and treatment clearly improved clinical signs. Age significantly affected the response to treatment in aggressive dogs; 75% of the cases responded with an improvement. The only minor and possibly treatment-related events observed were a short-lasting exacerbation of clinical signs of BPH (two dogs), increased weight gain (three dogs), and anxiety (three dogs) with one of these dogs developing a blunt coat. These results demonstrate the clinical efficacy and overall safety of the Gonazon implants.

Modeling the interaction of gametes and embryos with the maternal genital tract: From in vivo to in silico - Corrected Proof

A. Van Soom, L. Vandaele, L.J. Peelman, K. Goossens, A. Fazeli — 2010-01-25

Abstract: Understanding the complex interaction between gametes or embryos and the maternal genital tract requires the use of experimental models. The selection of the right model is an important task to undertake, and despite many new developments in this area, an ideal model system has not yet been developed. In this review article, we focus on how the most appropriate model species and model system can be selected, each with its particular advantages and disadvantages. Selection criteria need to be based on the evaluation of the aim of the experiment, the tools that are available to the scientist, and the ethics that are involved in working with particular animal species and model systems. Society and politics direct scientists to “Refine, Reduce, and Replace” the use of experimental animals, which means that the use of in vivo models is increasingly being discouraged. An in vivo model allows experimentation in the full biological environment of a living organism. In contrast with in vivo models, in vitro models are less complex and are abstracts of in vivo systems, leading often to results that are different from the in vivo situation. If an investigator could understand all the components of a complex biological system and re-create them as individual smaller models in a computer, he or she could create in silico models that would completely represent the complexity of in vivo models. We predict that in the future, in silico modeling will be the natural departure from in vivo, in situ, and in vitro modeling approaches. In addition to numerous advantages that this modeling approach can bring to studying maternal interaction with gametes and embryo, it is perhaps the only true alternative method to animal experimentation.

Rates of luteolysis and pregnancy in dairy cows after treatment with cloprostenol or dinoprost - Corrected Proof

J.S. Stevenson, A.P. Phatak — 2010-01-25

Abstract: Our objective was to determine whether rates of luteolysis or pregnancy differed in lactating dairy cows of known progesterone status and either known or unknown luteal status after either cloprostenol or dinoprost was injected as part of a timed-insemination program. In Experiment 1, 2358 lactating dairy cows in six herds were given two injections of PGF2α 14 d apart (Presynch), with the second injection given 12 to 14 d before the onset of a timed AI protocol (Ovsynch). Cows (n=1094) were inseminated when detected in estrus after the Presynch PGF2α injections. Cows not inseminated (n=1264) were enrolled in the Ovsynch protocol and assigned randomly to be treated with either cloprostenol or dinoprost as part of the timed-AI protocol. In cows having pretreatment concentrations of progesterone ≥1ng/mL and potentially having a functional corpus luteum (CL) responsive to cloprostenol (n=558) or dinoprost (n=519), dinoprost increased (P<0.05) luteal regression from 86.6 to 91.3%. Despite a significant increase in luteolysis, pregnancies per AI did not differ between luteolytic agents (dinoprost=37.8% and cloprostenol=36.7%). Fertility was improved in cows of both treatments having reduced concentrations of progesterone at 72h and in cows showing signs of estrus. In Experiment 2, an ovulation-resynchronization program was initiated with GnRH or saline in 427 previously inseminated lactating dairy cows of unknown pregnancy status in one herd. Seven days later, pregnancy was diagnosed and nonpregnant cows were blocked by number of CL and assigned randomly to be treated with cloprostenol or dinoprost. Compared with cloprostenol, dinoprost increased (P<0.05) luteal regression from 69.1 to 78.5%, regardless of the number of CL present or the total luteal volume per cow. Pregnancies per AI did not differ between dinoprost (32.8%) and cloprostenol (31.3%). Although dinoprost was more effective than cloprostenol at inducing luteolysis in lactating dairy cows exposed to an Ovsynch or ovulation-resynchronization protocol, resulting fertility did not differ between products.

The influence of washing Spanish ibex (Capra pyrenaica) sperm on the effects of cryopreservation in dependency of the photoperiod - Corrected Proof

M.A. Coloma, A. Toledano-Díaz, A. López-Sebastián, J. Santiago-Moreno — 2010-01-22

Abstract: Extenders containing low concentrations of egg yolk are recommended for cryopreserving ibex spermatozoa. However, the phylogenetic relationship of the Spanish ibex (Capra pyrenaica) with domestic goats suggests that phospholipases in the seminal plasma may have a negative effect on the response to freezing-thawing when egg yolk–based diluents are employed. The aim of the current work was to determine how seminal plasma removal from Spanish ibex semen, collected by electroejaculation over a period of 1 yr, affects its response to freezing-thawing. Semen was collected from six adult ibexes maintained in captivity. The negative effects of freezing-thawing on the quality of sperm motility and on the integrity of the acrosome and plasma membrane were more serious in the nonwashed semen samples than in those from which the seminal plasma had been removed (P<0.01, P<0.05, and P<0.05 respectively). The beneficial effect of removing the seminal plasma was particularly noticeable during the time of decreasing photoperiod. This suggests that ibex semen shows increased phospholipase activity during the rutting season.

New aspects of gamete transport, fertilization, and embryonic development in the oviduct gained by means of live cell imaging - Corrected Proof

S. Kölle, S. Reese, W. Kummer — 2010-01-18

Abstract: The integrity of gamete transport, fertilization, and early embryonic development in the oviduct are essential prerequisites for successful reproduction. Although the basic mechanisms of gamete transport, gamete interaction, and early embryogenesis are known in most mammals, the interactions between gametes and oviductal epithelium as well as the communication between the early embryo and the female reproductive tract remain to be elucidated. Recent techniques of live cell imaging such as digital videomicroscopy and confocal fluorescence microscopy are valuable tools that provide actual new insights into these interactions. By applying these techniques, the mechanisms of sperm transport, sperm storage, oocyte transport, gamete interaction, and early embryo-maternal crosstalk can be analyzed under in vivo or in situ conditions. Detailed knowledge of these very early and important processes creates the basis to develop new therapeutic concepts for subfertility and infertility and to improve the techniques of assisted reproduction. The current review will focus on a short description of recent techniques of live cell imaging in the reproductive tract followed by an overview of actual observations during the early events of reproduction.

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows - Corrected Proof

2010-01-18

Abstract: Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2×2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5±2.3 d postpartum, received a 3mg norgestomet ear implant sc, plus 3mg norgestomet and 5mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100μg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means±SEM, 1.53±0.1 vs. 0.48±0.1mm/d; P<0.0001), its diameter on Day 11 (11.4±0.6 vs. 9.3±0.7mm; P=0.03), as well as ovulation rate (80.8% vs. 50.0%, P=0.02), whereas GnRH improved the synchrony of ovulation (72.0±1.1 vs. 71.1±2.0h). In Experiment 2 (n=599 cows, 40 to 120 d postpartum), pregnancy rates differed (P=0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG+GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P=0.002; and 48.0% vs 37.6%, P=0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.

Endometrial biopsy: a valuable clinical and research tool in bovine reproduction - Corrected Proof

2010-01-18

Abstract: Studies of postpartum endometrial physiologic and immune mechanisms in cows are compromised by the difficulty in acquiring tissue of suitable quality and in sufficient quantity (Bos taurus). Endometrial biopsy sampling has attracted concern regarding potential animal ill-health and perturbed subsequent fertility. Here, we describe a method of endometrial biopsy that obtains high-quality tissue samples and does not compromise fertility. Using a Hauptner instrument, endometrial biopsies were taken at 15, 30, and 60 d postpartum from 13 mixed-breed beef cows. The effects of repeat biopsy on health (heart rate, respiration rate, color of mucous membranes, rectal temperature), onset of estrous cyclicity, and first service conception rate were monitored. Extensive daily clinical examinations revealed no signs of ill-health. All cows had resumed estrous cyclicity at 60 d postpartum. A conception rate of 77% was achieved after estrus synchronization and artificial insemination. Each biopsy yielded intact endometrial tissue and nucleic acid suitable for extensive histologic and molecular analysis, respectively. We conclude that when carried out appropriately, bovine endometrial biopsy is a safe and reliable technique for assessing postpartum uterine function or health.

Embryo and larva development in common dentex (Dentex dentex), a pelagophil teleost: The quantitative composition of egg-free amino acids and their interrelations - Corrected Proof

S-M. Samaee, E. Mente, A. Estévez, G. Giménez, F. Lahnsteiner — 2010-01-18

Abstract: Free amino acids (FAAs) play a key role in the physiology of marine teleosts (eggs, embryos, and larvae). However, the relationship between the egg FAAs content and the production of viable embryos and larvae (at different developmental stages) in batch spawner pelagophils has not yet comprehensively been investigated. Viable eggs of common dentex, Dentex dentex, were obtained from captive broodstocks. Egg wet weight (WW), dry weight (DW), and water content (%W) and viability parameters, or VPs (egg floating rate [FR], hatching rate [HR], and larval survival rate [SR] at days 0 to 5 posthatch) were determined for 45 egg batches. The egg batches were classified according to their HR magnitude. Twelve egg batches with the same WW, DW, and %W were taken from the same broodstock and at the same developmental stage to determine the qualitative and quantitative composition of FAAs. The total FAA (TFAA) content, glutamic acid (Glu), asparagine (Asn), glutamine (Gln), and arginine (Arg) were correlated with VPs. The Glu was significantly correlated with HR and SR at 0 day posthatch (dph), the Asn with SR at 1 dph, and the Gln and Arg with FR and HR. Of the 361 ratios made based on the absolute concentrations of FAAs, 24 ratios were correlated with VPs (P<0.005) through 42 simple regression models (R2=0.641 to 0.846). Of the 42 significant relationships found ∼10%, ∼28%, ∼12%, ∼30%, ∼8%, ∼4%, ∼2%, ∼2%, and ∼2% of the models show the relations of the egg FAAs ratios with FR, HR, SR at days 1 to 5 posthatch, and %W, respectively. A path coefficient in combination with a Pearson's correlation coefficient provided a series of statistical evidences to show the effects of the egg FAAs interrelations on the relationships found between quantitative composition of a FAA and a VP.

Silent ovulation, based on walking activity and milk progesterone concentrations, in Holstein cows housed in a free-stall barn - Corrected Proof

R.M.S.B.K. Ranasinghe, T. Nakao, K. Yamada, K. Koike — 2010-01-18

Abstract: The objectives of the current study were to determine the incidence of silent ovulation (based on walking activity and milk progesterone profiles), identify risk factors for silent ovulation, and investigate its impact on reproductive performance in high-yielding dairy cows in free-stall housing. Overall, 277 lactations in 161 Holstein Friesian cows from a commercial dairy herd in northern Japan were studied. Walking activity (measured with pedometers) >80% above the mean for the preceding 2 d was defined as estrus, whereas day of ovulation was estimated using milk progesterone concentrations. Ovulation not preceded by increased walking activity was considered silent ovulation; the incidence was 55.2%, 23.8%, 21.3%, and 10.5% at the first, second, third, and fourth ovulations postpartum, respectively. Moderate and high milk yield significantly increased the risk of silent ovulation at second (odds ratio [OR]=2.7 and 1.2; P=0.04) and third and/or fourth ovulations (OR=6.7 and 12.9; P=0.03). Based on survival analysis, silent ovulations at the first, second, third, and/or fourth ovulations were associated with 28% (hazard ratio [HR]=0.72), 55% (HR=0.45), and 47% (HR=0.53) reductions in pregnancy rate, respectively, and 41% (HR=0.59), 66% (HR=0.34), and 65% (HR=0.35) reductions in artificial insemination (AI) submission rate. Cows with at least one silent ovulation (with the exception of the first ovulation) had a longer interval from calving to first AI (72 vs. 54 d, P<0.001) and to achievement of pregnancy (133 vs. 80 d, P<0.001). In conclusion, approximately one third of the ovulations (based on milk progesterone concentrations) in Holstein cows within 90 d postpartum were silent. Silent ovulations at the second to fourth ovulations were associated with high milk yields and at all ovulations were associated with impaired reproductive performance.

Seasonal functional relevance of sperm characteristics in equine spermatozoa - Corrected Proof

S. Gamboa, A.S. Rodrigues, L. Henriques, C. Batista, J. Ramalho-Santos — 2010-01-18

Abstract: A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September–December, January–March, late March–May, and June–July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P<0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P<0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations.

Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys - Corrected Proof

F. Camillo, D. Panzani, C. Scollo, A. Rota, A. Crisci, I. Vannozzi, S. Balbo — 2010-01-18

Abstract: Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N=7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds.

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste - Corrected Proof

D. Zambelli, E. Iacono, R. Raccagni, B. Merlo — 2010-01-14

Abstract: An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro–matured cat oocytes. Cleavage rate was recorded 30h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P>0.05). Sperm membrane integrity was positively affected (P<0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P>0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6h after thawing (P>0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P>0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.

Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size - Corrected Proof

2010-01-14

Abstract: The objective of this study was to assess the reliability of luteal blood flow (LBF) as recorded by color Doppler sonography to monitor luteal function during the estrous cycle of dairy cows and to compare the results with that for the established criterion luteal size (LS) as determined by B-mode sonography. In total, 14 consecutive sonographic examinations were carried out in 10 synchronized lactating Holstein-Friesian cows (Bos taurus) on Days 4, 5, 6, 7, 8, 10, 12, 14, 16, –5, –4, –3, –2, –1 of the estrous cycle (Day 1=ovulation). Plasma progesterone concentrations in venous blood (P4) were quantified by enzyme immunoassay. Luteal size was determined by sonographic measurement of the maximal cross-sectional area of the corpus luteum (CL). Luteal blood supply was estimated by calculating the maximum colored area of the CL from power Doppler sonographic images. Luteal size doubled during the luteal growth phase (until Day 7) and remained at this level during the luteal static phase (Day 8 to 16) before decreasing rather slowly during luteal regression (Days –5 to –1). Luteal blood flow doubled during the growth phase, doubled furthermore during the static phase, and decreased rapidly during luteal regression. Thus, LBF values represented highly reliable predictors of luteal status. Luteal blood flow predicted reliably a P4>1.0 ng/mL by reaching only 35% of the maximal values, whereas LS had to exceed 60% of the maximal values to indicate reliably a functional CL. It is concluded that LBF reflected luteal function better than LS specifically during luteal regression.

Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency - Corrected Proof

D.B.B.P. Paris, T.A.E. Stout — 2010-01-14

Abstract: Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited potential for proliferation and differentiation in vitro, and do not appear to noticeably improve long-term functional repair. Embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome many of the limitations and be used to generate tissues of value for equine regenerative medicine. To date, six lines of putative ESCs have been described in the horse. All expressed stem cell–associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit pluripotency in vivo. Moreover, it is becoming clear that the markers used to characterize the putative ESCs were inadequate, primarily because studies in domestic species have revealed that they are not specific to ESCs or the pluripotent inner cell mass, but also because the function of most in the maintenance of pluripotency is not known. Future derivation and validation of equine embryonic or other pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing horse embryo.

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up - Corrected Proof

J.H.M. Viana, M.P. Palhao, L.G.B. Siqueira, J.F. Fonseca, L.S.A. Camargo — 2010-01-14

Abstract: The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n=10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2±0.3mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P<0.0001). There was a tendency (P=0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P<0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P<0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid - Corrected Proof

B. Agung, Y. Piao, D. Fuchimoto, S. Senbon, A. Onishi, T. Otoi, T. Nagai — 2010-01-11

Abstract: The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48h in pFF supplemented with (+) or without (−) FCs (5.2×106 cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O2. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O2 using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.

Microencapsulation of canine sperm and its preservation at 4°C - Corrected Proof

S. Shah, M. Nagano, Y. Yamashita, M. Hishinuma — 2010-01-07

Abstract: The objective of this study was to develop a preservation method for canine sperm using microencapsulation. Pooled ejaculates from three beagles (Canis familiaris) were extended in egg yolk Tris extender and were encapsulated in gel (alginate only) or polycation (poly-l-lysine membrane bound) microcapsules at 0.75% and 1.0% alginate concentration. In Experiment 1, characteristics of microcapsule and microencapsulated sperm were evaluated during chilling storage for 48h. Gel microcapsules at 0.75% alginate concentration had a teardrop-like structure with fragility, whereas those at 1.0% alginate had a solid spherical structure. In all groups, diameter of the microcapsules increased with duration of storage (P<0.05). Alginate concentration did not affect the sperm recovery rate from microcapsules. Total average recovery rate of sperm from polycation microcapsules was lower than that of gel microcapsules (P<0.05). Progressive motility of polycation microencapsulated sperm and unencapsulated sperm (control) was higher than that of the gel microencapsulated sperm, both at 0.75% and 1.0% alginate concentration (P<0.05), although viability of sperm was similar among the three groups. In Experiment 2, to evaluate the sperm longevity after chilling storage, sperm were microencapsulated in polycation microcapsules at 1.0% alginate concentration, stored at 4°C for 0, 1, 4, and 7 d, and then cultured at 38.5°C for 0, 6, and 24h. Progressive motility and viability of microencapsulated sperm were higher than those of unencapsulated spermatozoa at 0 to 24h of culture after 4 and 7 d of chilling storage (P<0.05). In conclusion, polycation microencapsulation at 1.0% alginate concentration can be successfully applied for chilling storage of canine sperm by maintaining motility and viability for up to 7 d.

Production of dairy goat embryos, by nuclear transfer, transgenic for human acid β-glucosidase - Corrected Proof

2010-01-06

Abstract: Expression of recombinant human lysosomal acid β-glucosidase (hGCase) by a transgenic animal bioreactor, using somatic cell nuclear transfer (SCNT), would decrease the cost of producing this product. The objective was to establish an effective procedure to prepare hGCase transgenic donor cells and nuclear transfer (NT) embryos to produce hGCase protein in the Saanen dairy goat mammary gland. A mammary-specific expression vector for hGCase was constructed and transfected into HC-11 mammary epithelial cells for bioactivity analysis in vitro; mRNA transcripts and hGCase protein were correctly expressed in transfected HC-11 cells. The hGCase gene was then introduced into fetal fibroblasts (from dairy goats) to prepare competent transgenic donor cells. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates and screened with PCR amplification and chromosomal counting (66.8%). Dairy goat cloned embryos were produced from these hGCase fetal cells by SCNT, the hGCase transgene was successfully detected in these embryos, and there were similar rates (P>0.05) of fusion (83.3% vs. 77.8%), cleavage (89.1% vs. 90.9%), and development to the morula/blastocyst stages (36.4% vs. 38.9%) between NT embryos using transgenic fetal fibroblasts and non-transfected control cells. Moreover, 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients; pregnancy was confirmed at 40 d in two goats. Therefore, we achieved functional expression of hGCase in mammary gland cells and normal development to Day 40 of cloned embryos carrying the hGCase gene.

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen - Corrected Proof

J.E. Larson, G.C. Lamb, B.J. Funnell, S. Bird, A. Martins, J.C. Rodgers — 2010-01-06

Abstract: This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1×106 sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n=32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15×106 sperm/straw; Conventional) or sexed semen (≥2.1×106 sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9±1.8 vs. 10.5±1.6), but the number of Grade 1 embryos was greater (P<0.01) for Conventional (4.3±0.8 vs. 2.3±0.7). Conversely, the mean number of unfertilized ova was greater (P<0.05) for Sexed (5.6±1.0 vs. 3.0±1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P<0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development - Corrected Proof

M.L. Mocé, A. Blasco, M.A. Santacreu — 2010-01-06

Abstract: The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72h of gestation. Morphologically normal embryos recovered at 72h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72h before transfer when fresh embryos were transferred and 60 to 63h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development - Corrected Proof

M.R. Tabandeh, A. Hosseini, M. Saeb, M. Kafi, S. Saeb — 2010-01-04

Abstract: Adiponectin is one of the most important, recently discovered adipocytokines that acts at various levels to control male and female fertility through central effects on the hypothalamus-pituitary axis or through peripheral effects on the ovary, uterus, and embryo. We studied simultaneous changes in the gene expression pattern of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in granulosa and theca cells, cumulus-oocyte complex, and in corpus luteum in healthy bovine (Bos tarus) follicles at different stages of development. The expression levels of adiponectin, AdipoR1, and AdipoR2 mRNA were lower (P<0.05) in granulosa and cumulus cells in comparison with that in theca cells and oocyte. In contrast with the oocyte, AdipoR1 in granulosa, theca, and luteal cells was expressed (P<0.05) more than AdipoR2. Adiponectin expression increased (P<0.05) in granulosa cells and in cumulus-oocyte complex during follicular development from small to large follicles. Opposite results were observed in theca cells. Expression of adiponectin was highest in the late stages of corpus luteum (CL) regression, whereas lower expression was recorded in active CL (P<0.05). AdipoR1 and AdipoR2 expression increased during the terminal follicular growth in granulosa and theca cells (P<0.05) and during the luteal phase progress in CL. There was positive correlation between adiponectin mRNA level in granulosa cells from large follicles and follicular fluid estradiol concentration (r=0.48, P<0.05) and negative correlation between adiponectin mRNA abundance in theca cells and follicular fluid progesterone concentration (r=–0.44, P<0.05). In conclusion, we found that the physiologic status of the ovary has significant effects on the natural expression patterns of adiponectin and its receptors in follicular and luteal cells of bovine ovary.

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua - Corrected Proof

I.A.E. Butts, M.K. Litvak, E.A. Trippel — 2010-01-04

Abstract: The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30sec post-activation) increased from 78.9±6.5 to 128.2±6.5μm/sec (mean±SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6±2.4 to 48.9±3.1μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6±4.2% in January to 40.5±4.4% in April; P<0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values<0.01). Seminal plasma osmolality and Na+ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P<0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P<0.001), whereas Ca2+ increased then decreased (P<0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe - Corrected Proof

M.W.H. Chowdhury, R.J. Scaramuzzi, C.P.D. Wheeler-Jones, M. Khalid — 2009-12-30

Abstract: Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n=5) or before (n=5), during (n=4), and after (n=4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP–labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5mm) and medium (2.5 to 4.0mm) follicles, but large follicles (>4.0mm) expressed higher mRNA and protein levels (all P<0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.

How to study placental vascular development? - Corrected Proof

F. Herr, N. Baal, R. Widmer-Teske, T. McKinnon, M. Zygmunt — 2009-12-28

Abstract: Both exogenous and endogenous factors during pregnancy may impact placental vascular development and cause different malformations of placental vessels. In humans, consequences of abnormal vascular development have been associated with different pregnancy-related pathologies ranging from miscarriage to intrauterine growth restriction or preeclampsia. Pregnancy-associated exposure to bacterial or viral infections or pharmacologic or toxic agents may also influence vascular development of the placenta and lead to preterm labor and delivery. Several steps of vascular adaptation on both the fetal and maternal side are necessary and include such events as uterine vasodilation, remodeling by extravillous trophoblast, as well as vasculogenesis and angiogenesis within the placenta. Ubiquitous as well as pregnancy-specific angiogenic factors are involved. Morphologic and stereologic approaches, as well as experiments in established laboratory animals, cannot be applied to large domestic animals or humans without hesitation. Thus, further studies into the different aspects of this process will require an appropriate in vitro model of placental vascular development. Reflecting the core of placental vascular development, the in vitro model should facilitate the interactions between trophoblast and stromal cells with endothelial progenitor cells. The effects of viral or bacterial infection as well as pharmacologic or toxic agents may be studied more closely in the process. This report reviews major aspects of vascular development in the placenta and describes the establishment of a three-dimensional in vitro model of human placental vascular development.

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro - Corrected Proof

J.A. Neira, D. Tainturier, M.A. Peña, J. Martal — 2009-12-25

Abstract: This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro–produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe - Corrected Proof

2009-12-25

Abstract: There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP2 and EP4, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100μg estradiol benzoate, and their cervices were collected 0, 24, and 48h thereafter to determine the expression of cervical COX-2, EP2, and EP4 mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP4 within 24h after treatment (P<0.05), whereas EP2 mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48h after treatment. The COX-2, EP2, and EP4 mRNA expression were greatest in the smooth muscle layers (P<0.05) and least in the luminal epithelium (P<0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP4 mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E2 and activation of the PGE2 receptors EP2 and EP4.

Effects of flaxseed dietary supplementation on sperm quality and on lipid composition of sperm subfractions and prostatic granules in rabbit - Corrected Proof

E. Mourvaki, R. Cardinali, A. Dal Bosco, L. Corazzi, C. Castellini — 2009-12-25

Abstract: Lipids are the main structural/functional components of the sperm, and their composition may undergo a series of modifications in relation to either physiologic events (capacitation and acrosome reaction) and/or diet. The goals of the current study were (1) to investigate whether a flaxseed (FS) dietary supplementation could affect the lipid and fatty acid profile of sperm subfractions and of prostatic granules (PGs) and (2) to evaluate the effects of dietary FS on rabbit buck semen quality. Accordingly, 20 adult New Zealand White rabbits were fed ad libitum a control diet (CO) or a diet supplemented with 5% extruded FS. Integration of diet with FS, as a consequence of the linolenic acid (C18:3n-3; LNA; 56%), increased the dietary n-3/n-6 ratio and resulted in a substantial rearrangement of sperm fatty acid composition at the subcellular level, mainly of polyunsaturated fatty acid (PUFA)n-3 (8.3% vs. 14.3%, P<0.05). The lipid and fatty acid profiles of sperm tail membrane were the most affected, undergoing the following significant changes: (1) a reduction by half of linoleic acid (C18:2n-6; LA) and docosapentaenoic acid (22:5n-6; DPA), and a reduction of cholesterol (−70%); (2) a concomitant increase of LNA (+65%), docosahexaenoic acid (22:6n-3; DHA; +83%), and of oleic acid (C18:1n-9, +61%). As a consequence, the sperm of FS-fed rabbits had a twice higher n-3/n-6 ratio and phospholipid/cholesterol ratio compared with the control sperm. These changes might have been on the basis of the higher responsiveness to hypo-osmotic solution and, hence, the higher sperm track speed observed for the FS group. Also, the membrane integrity and viability of the LNA-enriched sperm were both improved. On the other hand, the presence of lignans in FS might have accounted for the reduction of sperm cholesterol in the semen of FS-treated rabbits. The responsiveness of sperm to acrosome reaction was not affected by the dietary treatment probably due to supranutritional level of vitamin E and to the higher number of PGs, which are known to play a key role in sperm capacitation. In conclusion, our data showed for the first time that the integration of FS into the rabbit diet may improve sperm quality by modifying the sperm lipid composition and that the sperm subfractions and the PGs respond differently to the FS-induced lipid manipulation.

Ovarian antral follicular dynamics in sheep revisited: Comparison among estrous cycles with three or four follicular waves - Corrected Proof

2009-12-25

Abstract: In this study, the characteristics of ovarian follicular waves and patterns of serum concentrations of follicle-stimulating hormone (FSH), estradiol, and progesterone were compared between cycles with three (n=9) or four (n=10) follicular waves in Western White Face (WWF) ewes (Ovis aries). Transrectal ultrasonography and blood sampling were performed daily during one cycle. Estrous cycles were 17.11±0.3 and 17.20±0.2 d long in cycles with three and four waves, respectively (P>0.05). The first interwave interval and the interval from the emergence of the final wave to the day of ovulation were longer in cycles with three waves compared with those in cycles with four waves (P<0.05). The growth phase (5.1±0.5 vs. 3.1±0.4 d) and life span (5.67±0.3 vs. 4.3±0.3 d) of the largest follicle growing in the last or ovulatory wave was longer in cycles with three waves compared with that in cycles with four waves (P<0.05). The maximum diameter of the largest follicle was greater in the first wave and the ovulatory wave compared with that in other waves of the cycle (P<0.05). The regression phase of the largest follicle growing in the first wave was longer in cycles with three waves compared with that in cycles with four waves (4.44±0.4 vs. 3.4±0.4 d; P<0.05). The length of the life span, regression phase, and, although not significant in every case, FSH peak concentration and amplitude decreased across the cycle (P<0.05). We concluded that estrous cycles with three or four follicular waves were confined within the same length of cycle in WWF ewes. In this study, there were no apparent endocrine or follicular characteristics that could explain the regulation of the different number of follicular waves (three vs. four) during cycles of similar length.

Is Doublesynch protocol a new alternative for timed artificial insemination in anestrous dairy cows - Corrected Proof

Ö.A. Öztürk, Ü. Cirit, A. Baran, K. Ak — 2009-12-21

Abstract: This is the very first report that suggests high pregnancy rates can be obtained with use of the Doublesynch protocol in anestrous dairy cows. Recently, a new synchronization method has been developed (Doublesynch) that resulted in synchronized ovulations both after the first and second gonadotropin-releasing hormone (GnRH) treatments. It was suggested that this protocol has the potential to increase the pregnancy rates in primiparous dairy cows. The aim of the current study was to confirm the success of the Doublesynch protocol and further to investigate the effect of this method on pregnancy rates in anestrous cows. Lactating primiparous Holstein (Bos taurus) cows (n=165) between 60 and 172 d postpartum were monitored twice with 10-d intervals (on Days –10 and 0) by ultrasonography, and blood samples were collected. Cows were classified as anestrous if both blood samples had progesterone (P4) concentration <1 ng/mL and as cyclic if at least one of the two samples had P4 concentration ≥1 ng/mL. Cyclic cows were classified again as cyclic-high P4 (having an active corpus luteum) if the second blood samples had P4 concentrations ≥1 ng/mL and as cyclic-low P4 if P4 concentrations were <1 ng/mL on Day 0. Then, the cows classified as anestrous (n=51), cyclic-high P4 (n=51), or cyclic-low P4 (n=63) were put into two treatment groups (Ovsynch or Doublesynch) randomly to establish six groups. Cows in the Ovsynch group were administered a GnRH (lecirelin 50μg, im) on Day 0, PGF (Prostaglandin F2 alpha, D-cloprostenol 0.150mg, im) on Day 7, and a second dose of GnRH 48h later. Cows in the Doublesynch group were administered a PGF on Day 0, GnRH on Day 2, a second PGF on Day 9, and a second GnRH on Day 11. Timed artificial insemination (TAI) was performed 16 to 20h after the second GnRH in both treatment groups. Pregnancy diagnosis was conducted (by ultrasonography) 45±5 d after TAI. In anestrous cows and those with high and low progesterone concentration at treatment onset, Doublesynch treatment led to markedly increased pregnancy rates with respect to Ovsynch treatment (P<0.05). On the overall analysis of data, it was revealed that the Doublesynch method increased pregnancy rates by 43 percentage units (29.8% vs. 72.8%, P<0.0001) in relation to Ovsynch. Pregnancy rates of cows having small, medium, or large follicles at the day of second GnRH administration were similar in the Doublesynch group (70.4%, 85.2%, and 63.0%, respectively; P>0.05), whereas pregnancy rates reduced dramatically as follicle size increased in the Ovsynch group, particularly in cows with follicles greater than 16mm (45.5%, 28.1%, and 5.3%, respectively; P<0.05). Our results confirm and support observations that the Doublesynch protocol increases the pregnancy rates in postpartum primiparous cows as reported previously. Our data also demonstrate that the Doublesynch method increases the pregnancy rates in anestrous cows. Thus, these data suggest that the Doublesynch protocol can be used to obtain satisfactory pregnancy rates after TAI in both anestrous and cycling primiparous dairy cows regardless of stage of estrous cycle.

A diet supplemented with l-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase - Corrected Proof

M. Yeste, S. Sancho, M. Briz, E. Pinart, E. Bussalleu, S. Bonet — 2009-12-18

Abstract: It has been reported that a diet supplemented with l-carnitine can improve sperm quality in some mammalian species. Against this background, the current study seeks to determine the effects of feeding l-carnitine (625mg·day−1) on boar semen characteristics (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) in three different porcine breeds (Sus domesticus) (Piétrain, Duroc, and Large White) exposed to natural environmental changes in temperature and photoperiod over a 20-wk period (February to July 2007). One hundred twenty boars (40 per breed) were randomly separated into two groups (60 boars each): the first (20 boars per breed) was fed a control diet and the second (also 20 males per breed) the same diet supplemented with l-carnitine (625mg·day−1). Whereas the l-carnitine supplement did not affect ejaculate volume, concentration, motility, viability, or the osmotic resistance of spermatozoa, it did improve sperm morphology in Piétrain boars by reducing the percentage of immature spermatozoa when the temperature and the photoperiod increased. Conversely, no effect on sperm morphology from supplementing feed with l-carnitine was observed in both Duroc and Large White breeds. We can therefore conclude that the addition of l-carnitine to the diet of males may maintain the level of normal sperm morphology in Piétrain boars when a drop in sperm quality occurs (due to increases in photoperiod and temperature), without affecting the other sperm quality parameters.

Respiratory and cardiovascular effects of doxapram and theophylline for the treatment of asphyxia in neonatal calves - Corrected Proof

U. Bleul, B. Bircher, R.S. Jud, A.P.N. Kutter — 2009-12-18

Abstract: Respiratory stimulants are widely used in asphyxic neonatal calves despite a lack of data about their effectiveness and indications of possible side effects. The effect of doxapram and theophylline on respiratory, cardiovascular, and acid-base variables was investigated in 10 healthy neonatal calves (Bos Taurus). A venous, a peripheral arterial, and a pulmonary arterial catheter were placed, and central venous, pulmonary, and systemic blood pressures and cardiac output were measured using thermodilution technique. Doxapram, but not theophylline, led to an immediate increase in respiratory rate (P ≤ 0.01). The arterial pCO2 decreased to 27.1±4.7mm Hg within 30sec after doxapram administration and to 46.3±5.8mm Hg within 120min after theophylline administration (P<0.0001). The systolic pulmonary pressure increased from 70±8mm Hg (mean±SD) to 93±19mm Hg within 30sec after doxapram, but decreased after theophylline. The pulmonary vascular resistance also increased after doxapram and decreased after theophylline (P<0.01). Doxapram had a more pronounced and faster effect on respiratory rate and elimination of CO2 than theophylline. Doxapram, but not theophylline, is indicated for treatment of postnatal asphyxia in calves, but there are potential cardiovascular side effects.

Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity - Corrected Proof

2009-12-17

Abstract: In an attempt to evaluate the protective effect of a lipid-soluble antioxidant (butylated hydroxytoluene; BHT), semen from four dogs (Canis familiaris) was frozen in two different extenders (Uppsala or INRA-96 plus glycerol) with or without 1mM BHT. Sperm membrane integrity using flow cytometry and motility using a computerized system were evaluated in each experimental group. The Uppsala extender was superior in all aspects of sperm function. The percentage of sperm membranes was significantly higher in semen samples frozen in presence of BHT. Our results suggest that the Uppsala extender can be improved with the addition of BHT.

Beyond the mouse model: Using Drosophila as a model for sperm interaction with the female reproductive tract - Corrected Proof

Y. Heifetz, P.K. Rivlin — 2009-12-16

Abstract: Although the fruit fly, Drosophila melanogaster, has emerged as a model system for human disease, its potential as a model for mammalian reproductive biology has not been fully exploited. Here we describe how Drosophila can be used to study the interactions between sperm and the female reproductive tract. Like many insects, Drosophila has two types of sperm storage organs, the spermatheca and seminal receptacle, whose ducts arise from the uterine wall. The spermatheca duct ends in a capsule-like structure surrounded by a layer of gland cells. In contrast, the seminal receptacle is a slender, blind-ended tubule. Recent studies suggest that the spermatheca is specialized for long-term storage, as well as sperm maturation, whereas the receptacle functions in short-term sperm storage. Here we discuss recent molecular and morphological analyses that highlight possible themes of gamete interaction with the female reproductive tract and draw comparison of sperm storage organ design in Drosophila and other animals, particularly mammals. Furthermore, we discuss how the study of multiple sperm storage organ types in Drosophila may help us identify factors essential for sperm viability and, moreover, factors that promote long-term sperm survivorship.

Culling intervals and culling risks in four stages of the reproductive life of first service and reserviced female pigs in commercial herds - Corrected Proof

Y. Sasaki, Y. Koketsu — 2009-12-14

Abstract: The objectives of this study were to measure culling intervals and culling risks in the four stages of the reproductive life of female pigs and to compare culling intervals between the number of services and between herd groups, based on herd productivity. We also compared survival patterns of females pigs between these herd groups. Our data set included lifetime records of 52,792 females born between 2001 and 2004 in 101 commercial herds. Two herd groups were selected on the basis of the upper 25th percentile of pigs weaned per mated female per 5 yr between 2002 and 2006, namely the high-performing herds, and ordinary herds. Culled females were also allocated into four groups based on the stages of their reproductive life when culled: unmated gilts, mated gilts, unmated sows, and mated sows. Culling intervals in unmated gilts and mated gilts were defined as the number of days from birth to culling and from first mating to culling, respectively. Culling intervals in unmated sows and mated sows were the number of days from weaning to culling. The number of services was categorized into two groups: first service and reservice groups. Multilevel linear mixed-effects models and survival analysis were performed. Culling intervals (±SEM) in unmated gilts, mated gilts, unmated sows, and mated sows were 302.9±1.16, 98.4±0.92, 14.3±0.12, and 89.6±0.42 d, respectively. Culling risks in the four groups were 5.6%, 7.1%, 58.0%, and 29.3%, respectively. In unmated gilts, mated gilts, and mated sows, the culling intervals in the high-performing herds were 43.0, 18.9, and 16.0 d shorter than those in ordinary herds, respectively (P<0.05), but no difference was found between the herd groups for the culling interval of unmated sows. For mated sows in the reservice group, culling intervals of high-performing herds were ≥13.7 d shorter than those of the ordinary herds (P<0.05), but for mated sows in the first service group, there was no difference in the culling interval between the herd groups. The culling hazard from 8 wk postweaning for mated sows in high-performing herds increased more rapidly than that in ordinary herds. In conclusion, to reduce culling intervals and improve herd productivity, we recommend implementing a strict culling policy for mated gilts and mated sows, especially reserviced females.

Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral (Echinopora spp.) oocytes - Corrected Proof

S. Tsai, E. Spikings, F.W. Kuo, N.C. Lin, C. Lin — 2009-12-14

Abstract: The objective was to examine the effects of cryoprotectants on oocytes of hard corals (Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0M) for 20min at room temperature (25°C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA)+propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay. Both FDA+PI staining and ATP assay indicated that cryoprotectant toxicity to oocytes increased in the order methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG). The no observed effect concentrations for Echinopora spp. oocytes were 1.0, 0.5, 0.25, and 0.25M for methanol, DMSO, PG, and EG, respectively, when assessed with FDA+PI. The ATP assay was more sensitive than FDA+PI staining (P<0.05). Oocyte viability after 1.0M methanol, DMSO, EG, or PG treatment for 20min at room temperature assessed with FDA+PI tests and ATP assay were 88.9±3.1% and 72.2±4.4%, 66.2±5.0% and 23.2±4.9%, 58.9±5.4% and 1.1±0.7%, and 49.1±5.1% and 0.9±0.5%, respectively. We inferred that the ATP assay was a valuable measure of cellular injury after cryoprotectant incubation. The results of this study provided a basis for development of protocols to cryopreserve coral oocytes.

Semen characteristics of genetically identical male cats cloned via somatic cell nucleus transfer - Corrected Proof

E.G. Choi, Y.S. Lee, S.J. Cho, J.T. Jeon, K.W. Cho, I.K. Kong — 2009-12-14

Abstract: We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (× 106/mL) of Cat A (512±140, range 368 to 685) was significantly higher, whereas that of Cat C (335±92, range 274 to 469) was significantly lower than that of Cloned B (459±159, range 336 to 510) and control cats (680±452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage=74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats.

Canine embryonic stem cells: State of the art - Corrected Proof

M.R. Schneider, E. Wolf, J. Braun, H-J. Kolb, H. Adler — 2009-12-07

Abstract: Embryonic stem cells (ESCs) are permanent cell lines that can be maintained in a pluripotent, undifferentiated state. Appropriate environmental stimuli can cause them to differentiate into cell types of all three germ layers both in vitro and in vivo. Embryonic stem cells bear many opportunities for clinical applications in tissue engineering and regenerative medicine. Whereas most of our knowledge on the biology and technology of ESCs is derived from studies with mouse cells, large animal models mimicking important aspects of human anatomy, physiology, and pathology more closely than mouse models are urgently needed for studies evaluating the safety and efficacy of cell therapies. The dog is an excellent model for studying human diseases, and the availability of canine ESCs would open new possibilities for this model in biomedical research. In addition, canine ESCs could be useful for the development of cell-based approaches for the treatment of dogs. Here, we discuss the features of recently reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.

In vitro systems for intercepting early embryo-maternal cross-talk in the bovine oviduct - Corrected Proof

S.E. Ulbrich, K. Zitta, S. Hiendleder, E. Wolf — 2009-12-07

Abstract: A comprehensive understanding of the complex embryo-maternal interactions during the preimplantation period requires the analysis of very early stages of pregnancy. These are difficult to assess in vivo due to the small size of the embryo exerting local paracrine effects. Specifically designed experiments and holistic transcriptome and proteome analyses to address the early embryo-maternal cross-talk in the oviduct require sufficient numbers of well-defined cells in a standardized experimental environment. The pronounced estrous cycle–dependent changes in gene expression and morphology of bovine oviduct epithelial cells (BOECs) clearly show that a precise definition of the stage of estrous cycle is essential for obtaining a well-defined homogenous population of functional cells. The number of intact cells isolated from individual ampullae by solely mechanical means was 10-fold higher than previously reported cell yields after enzymatic treatment, and the purity was comparable. Bovine oviduct epithelial cells have been cultured as monolayers or in suspension. Proliferating cells grown in monolayers dedifferentiated, with a concomitant loss of important morphologic characteristics. After several days in culture, BOECs in monolayers are less likely to mimic the oviduct environment in vivo than BOEC vesicles formed of epithelial sheets in short-term suspension culture. A 24-h culture system for BOECs isolated on Day 3.5 of the estrous cycle showed excellent preservation of morphologic criteria, marker gene expression, and hormone responsiveness. The short-term BOEC culture system provides well-defined and functional BOECs in sufficient quantities for studies of early embryo-maternal interactions in experiments that mimic the environment in the oviduct in vivo.