- Generation of α‐1,3‐Galactosyltransferase‐Deficient
Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in
of a Small Mutated Sequence and a Targeted Toxin‐Based Selection
- Authors: M Sato; A Kagoshima, I Saitoh, E Inada, K Miyoshi, M Ohtsuka, S Nakamura, T Sakurai, S Watanabe
Abstract: The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α‐gal epitope synthesized by α‐1,3‐galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig‐to‐human xenotransplantation. We here employed the CRISPR/Cas9 system‐mediated knock‐in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800‐bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α‐gal epitope expression, we successfully obtained several knock‐in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9‐mediated HR to knock‐in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.
- Therapeutic Ultrasound as a Potential Male Dog Contraceptive:
Determination of the Most Effective Application Protocol
- Authors: R Leoci; G Aiudi, F Silvestre, EA Lissner, F Marino, GM Lacalandra
Abstract: Ultrasound is one of the most promising forms of non‐invasive contraception and has been studied in several animal models. The objective of the current investigation was to determine the most practical and effective application protocol for dog sterilization. A total of 100 dogs were divided into five equal groups. Group A received 5‐min applications three times performed at 48‐hr intervals and covering the entire testicular area at frequency of 1 MHz; Group B received 5‐min applications three times performed at 48‐hr intervals over the dorso‐cranial area of the testis at frequency of 3 MHz; Group C received three sequential 5‐min applications (at 5‐min intervals between applications) covering the entire testicular area at frequency of 1 MHz; Group D received 15‐min applications two times performed at 48‐hr intervals and covering the entire testicular area at frequency of 1 MHz. The experimental groups' ultrasound had an intensity of 1.5W/cm2. The Control Group had the same procedure as Group A, but with the transducer switched‐off. Dogs were surgically castrated 40 days following the treatment for histological examination. Azoospermia, testicular volume reduction and apparently irreversible testicular damage were achieved by Group A. No effects were noticed in the other groups. Testosterone levels remained within physiological range with all application protocols. A regimen of three applications of ultrasound at 1 MHz, and 1.5 W/cm2, lasting 5 min with an interval of 48 h was effective as permanent sterilization in the dog without hormonal impact.
- Reproductive and Metabolic Responses of Early‐lactating Dairy Cows
Fed Different Dietary Protein Sources
- Authors: V Tufarelli; GM Lacalandra, V Laudadio
Abstract: Optimal reproduction is very closely tied with optimal nutrition, and early‐lactation diets in cows are critical to successful reproduction and monitoring is important. To evaluate the effects of different dietary protein sources on metabolic parameters and reproductive activity, a total of 36 Italian Friesian early‐lactating dairy cows were assigned for 16 weeks to three dietary treatments as follow: the control diet contained soya bean meal (SBM) as the main protein source, whereas the experimental diets contained faba bean (FB) or pea seeds (PS) as alternative protein sources. Diets were formulated to be isocaloric and isonitrogenous. Cow blood samples were collected, and plasma were analysed for metabolites, biological enzymes, β‐hydroxybutyrate (BHBA) and non‐esterified fatty acids (NEFA). Feeding alternative protein sources had no effects on most metabolic blood profile, except for blood cholesterol, triglycerides and urea. Results from reproductive parameters indicated that cows fed FB diet had a lower insemination index, but a shorter calving to conception period and an improved conception rate and artificial insemination outcome, when compared to cows fed SBM or PS diets. It can be concluded that replacing conventional dietary SBM with alternative protein sources, especially FB, resulted in improved reproductive performances and metabolic parameters in early‐lactating dairy cows.
- Cryopreservation and In Vitro culture of Preimplantation Embryos in
Djungarian Hamster (Phodopus sungorus)
- Authors: EYu Brusentsev; TO Abramova, IN Rozhkova, TN Igonina, VA Naprimerov, NYu Feoktistova, SYa Amstislavsky
Abstract: Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four‐cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third‐day post coitum and frozen in 0.25‐ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non‐permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non‐frozen four‐cell embryos developed up to the morula stage in rat one‐cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24‐h period of culture. The rate of embryo's surviving the freezing–thawing procedures, as estimated by light microscopy, was 60.7–68.8%. After 24‐h culturing in R1ECM, 64.7% of frozen–thawed four‐cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM‐CSF (2 ng/ml) improved the rate of Djungarian hamster frozen–thawed embryo development: 100% of the four‐cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen–thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.
- Derivation of Canine Induced Pluripotent Stem Cells
- Authors: AEG Baird; T Barsby, DJ Guest
Abstract: Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.
- Doppler and Contrast‐Enhanced Ultrasonography of Testicles in Adult
- Authors: MBS Brito; MAR Feliciano, LN Coutinho, RR Uscategui, APR Simões, MC Maronezi, VT Almeida, RM Crivelaro, B Gasser, L Pavan, WR Russiano
Abstract: The objective was to characterize the vascular patterns of testicular blood flow of adult cats, measuring the systolic velocity (SV), diastolic velocity (DV), resistance index (RI), gate time (wash‐in) peak enhancement and output time (wash‐out) of the contrast and addition of tissue fill characteristics. Forty‐five adult cats were selected, and the echotexture, echogenicity, size, contours and margins of testicles were assessed via ultrasound. By Doppler were evaluated the blood flow and determined of vascular index in testicular artery (SV, DV and RI) and via contrast‐enhanced ultrasonography determine the time for phases: wash‐in, wash‐out and peak enhancement. Sonographic findings presented normal. Testicular artery was observed in the spermatic cord with tortuous patter and showed monophasic‐patterned waves and low vascular resistance and with systolic peak evident. Values of indices vascular were as follows: SV = 6.73 cm/s, DV = 2.8 cm/s and RI = 0.54 for left testicles; and SV = 6.23 cm/s, DV = 2.77 cm/s and RI = 0.53 for right testicles. Contrast filled the subcapsular vascular structures and after a few seconds, a homogeneous moderate enhancement of the parenchyma, with parenchymal vessels still distinguishable and after the peak phase, a rapid homogeneous decrease in echogenicity. Values of time for contrast‐enhanced ultrasonography were as follows: wash‐in = 8.78 s, peak enhancement = 21.62 s and wash‐out = 75.36 for left testicles; and wash‐in = 10.76 s, peak enhancement = 21.50 s and wash‐out = 81.81 for right testicles. Doppler and contrast‐enhanced ultrasonography of the testicles in healthy adult cats was easily implemented and may provide baseline data for this organ to allow the use of these techniques as a diagnostic tool for evaluating testicular abnormalities in sick cats.
- Evidence of Spontaneous Recovery of Granulosa‐Theca Cell Tumour in a
Heifer: A Retrospective Report
- Authors: H El‐Sheikh Ali; G Kitahara, S Torisu, K Nibe, Y Kaneko, Y Hidaka, T Osawa
Abstract: A 14‐month‐old Japanese Black heifer was evaluated on Day 0 (D 0) for enlargement of the right ovary (RO). Transrectal ultrasonography (TRUS) revealed that the RO was markedly enlarged and multicystic, while the left ovary (LO) was small and inactive. The presumptive diagnosis was granulosa‐theca cell tumour (GTCT), which was confirmed by markedly elevated plasma anti‐Müllerian hormone (AMH) of 4.42 ng/ml. Therefore, ovariectomy of the RO was the treatment of choice. The heifer was checked by TRUS and blood sampling on D 42, D 63 and immediately before ovariectomy on D 85. On D 42, TRUS did not show marked changes in either ovary in comparison with D 0. However, on D 63, the RO had transformed into a single cyst, and on D 85, the LO had resumed cyclic activity. The RO was extracted on D 85 by hand‐assisted laparoscopic ovariectomy to allow better control. Unexpectedly, histopathology revealed the lesion to be a fluid‐filled cystic structure, with no neoplastic proliferation of follicular epithelium that would indicate GTCT. The wall of the cystic structure consisted of collagen fibres and a few degenerated granulosa cells. The retrospective hormonal analysis revealed that the AMH concentrations had markedly dropped on D 63 and 85, which coincided with resumption of cyclicity in the LO. These findings suggest that the GTCT had self‐cured and transformed into a cyst‐like structure. The heifer then received an oestrous synchronization regime on D 105, was artificially inseminated on D 115 and became pregnant.
- Application of an ELISA Milk Pregnancy Test in Beef Cows
- Authors: JN Roberts; TM Byrem, DL Grooms
Abstract: Pregnancy‐associated glycoproteins (PAG) are secreted by the binucleate giant cells of the ruminant placenta and enter maternal circulation at the time of placental attachment. The IDEXX Milk Pregnancy Test (IDEXX, Westbrook, ME) detects a subset of PAG in milk. Although designed as a management tool for dairy cows, there is potential for using the milk PAG test in beef cows. Our objective was to compare the performance of the milk PAG ELISA with a gold standard method for pregnancy diagnosis and determine the agreement between milk and serum PAG analysis in lactating beef cows. Angus and Angus‐crossed cows (n = 332) from two Michigan beef herds were enrolled in this study. Cows were subjected either to timed artificial insemination followed by exposure to a bull or exclusively exposed to a bull. The bulls and cows were separated 30 days prior to examination. Serum and milk samples were collected and submitted within 24 h of collection to a commercial laboratory for PAG analysis using the IDEXX Milk Pregnancy Assay (milk) and the IDEXX Bovine Pregnancy Assay (serum). Concurrently with milk and serum collection, each cow was examined transrectally by palpation or ultrasonography. When compared to transrectal examination, the performance (and 95% confidence intervals) of the milk PAG ELISA was sensitivity of 99.7% (99.0–100.0%) and specificity of 80.8% (65.6–95.9%). The lower specificity is likely due to the low prevalence (9.9%) of open cows (n = 30) in the herds examined. Of the 332 cows examined, 1.8% (n = 6) were classified as rechecks using the milk PAG ELISA. Results of the milk and serum PAG ELISA were in high agreement (kappa coefficient = 0.91). The milk PAG ELISA was accurate in predicting pregnancy status using milk collected from beef cattle between days 37 and 125 post‐insemination and may be useful for aiding management decisions in beef herds.
- Endogenous Progesterone Concentrations Affect Progesterone Release from
Intravaginal Devices Used for Oestrous Synchronization in Cattle
- Authors: HL Neri; MP Palhao, DS Costa, JHM Viana, CAC Fernandes
Abstract: Intravaginal progesterone‐releasing devices are largely used both as contraceptives in humans and as a component of oestrous synchronization protocols in cattle. To reduce costs in large‐scale timed artificial insemination, the reuse of these releasing devices is common. Passive hormone diffusion, however, depends on the concentration gradient, which could affect the amount of residual progesterone present in these devices after a first use. To evaluate the effect of the presence of a corpus luteum in the release of progesterone from intravaginal devices, three synchronization protocols were designed to simulate the effects of inserting the device in the early dioestrus, late dioestrus or anoestrus. Holstein‐Zebu cross‐bred heifers were randomly allocated into one of these three treatments, and a series of blood samples was taken to evaluate the plasma progesterone concentrations. After 8 days, the intravaginal devices were removed and underwent a previously validated alcoholic extraction technique to measure the residual progesterone. Non‐used devices were used as controls. As expected, the simultaneous presence of the intravaginal device and a corpus luteum resulted in increased plasma progesterone concentrations. Conversely, the amount of residual progesterone in the devices after use was inversely proportional to the plasma progesterone concentration. These results demonstrate that the release rate of progesterone from intravaginal devices is affected by the endogenous concentration of this hormone; consequently, the strategy for reuse should account for the category and expected luteal cyclic activity of the animals undergoing synchronization protocols.
- Involvement of PAPP‐A and IGFR1 in Cystic Ovarian Disease in Cattle
- Authors: FM Rodríguez; M Colombero, AN Amweg, E Huber, NC Gareis, NR Salvetti, HH Ortega, F Rey
Abstract: Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors, such as members of the insulin‐like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy‐associated plasma protein A (PAPP‐A). The aim of this study was to determine IGFR1 and PAPP‐A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP‐A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP‐A present in the follicular fluid from these follicles showed no differences. Although no PAPP‐A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.
- Cleaved PARP‐1, an Apoptotic Marker, can be Detected in Ram
- Authors: A Casao; M Mata‐Campuzano, L Ordás, JA Cebrián‐Pérez, T Muiño‐Blanco, F Martínez‐Pastor
Abstract: The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly‐ADP‐ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly‐ADP‐ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm) or betulinic acid (200 μm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p
- The Impact of Reproductive Technologies on Stallion Mitochondrial Function
- Authors: FJ Peña; M Plaza Davila, BA Ball, EL Squires, P Martin Muñoz, C Ortega Ferrusola, C Balao da Silva
Abstract: The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen–thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.
- Expression of miRNAs in Bull Spermatozoa Correlates with Fertility Rates
- Authors: M Fagerlind; H Stålhammar, B Olsson, K Klinga‐Levan
Abstract: Bull spermatozoa are rich in active miRNAs, and it has been shown that specific spermborne miRNAs can be linked to fertility. Thus, expression profiling of spermatozoa could be helpful for understanding male fertility and the ability of spermatozoa to initiate and sustain zygotic, embryonic and foetal development. Herein we hypothesized that bulls with moderate to high fertility can be identified by differences in amounts of certain miRNAs between their ejaculates. RNA samples from spermatozoa of eight brother pairs (one bull with high and one with moderate NRR in each pair) of the Holstein breed were prepared. miRNA was isolated, and the expression of 178 miRNAs was determined by RT‐qPCR. Important findings were that highly expressed miRNAs, not linked to NRR status, were identified in the bull sperm samples, which indicate that these miRNAs have an important role in early embryogenesis. A large fraction of the targets genes were phosphoproteins and genes involved in the regulation of transcription. Seven miRNAs (mir‐502‐5p, mir‐1249, mir‐320a, mir‐34c‐3p, mir‐19b‐3p, mir‐27a‐5p and mir‐148b‐3p) were differentially expressed between bulls with moderate and high NRR with a strong tendency towards a higher expression of miRNAs in bulls with moderate fertility. Thus, bulls with a moderate NRR negatively regulate the expression of protein‐coding genes, which leads to problems during the pregnancy.
- The Effect of Seasonal Thermal Stress on Lipid Mobilisation, Antioxidant
Status and Reproductive Performance in Dairy Cows
- Authors: R Turk; O Podpečan, J Mrkun, Z Flegar‐Meštrić, S Perkov, P Zrimšek
Abstract: Heat stress is a major factor contributing to low fertility of dairy cows with a great economic impact in dairy industry. Heat‐stressed dairy cows usually have reduced nutrient intake, resulting in a higher degree of negative energy balance (NEB). The aim of this study was to investigate the seasonal thermal effect on lipid metabolism, antioxidant activity and reproductive performance in dairy cows. Thirty‐two healthy dairy heifers were included in the study. According to the ambient temperature, animals were divided into two groups: winter (N = 14) and summer season (N = 18). Metabolic parameters, paraoxonase‐1 (PON1) activity and total antioxidant status (TAS) were monitored at the time of insemination (basal values) and from 1 week before until 8 weeks after calving. Number of services per conception and calving‐to‐conception (CC) interval were calculated from the farm recording data. Serum triglyceride, non‐esterified fatty acids (NEFA) and beta‐hydroxybutyrate (BHB) concentrations were significantly increased after calving in summer compared to winter, indicating higher degree of NEB in cows during summer. PON1 activity was significantly decreased after calving in both summer and winter group. TAS concentration was significantly lower in summer than that in winter. A significantly higher number of services were needed for conception in summer compared to winter, and CC interval was significantly longer in summer than that in winter as well. Additionally, reproductive performance significantly correlated with the severity of NEB, suggesting that lipid mobilization and lower antioxidant status contributed to poor reproduction ability in dairy cows during hot months.
- Comparative Development of Embryonic Age by Organogenesis in Domestic Dogs
- Authors: NCG Pieri; AF Souza, JB Casals, KCS Roballo, CE Ambrósio, DS Martins
Abstract: The precise determination of the embryonic chronology is very important in reproductive biotechnologies, especially in estimating embryonic age. Thus, there is a need for greater knowledge and standardization for determining the chronology of embryonic development and functional morphology. We describe aspects of embryonic development in two domestic carnivores to add knowledge about organ peculiarities and for application in veterinary practice, in prenatal development and in the biotechnology fields. We found that the development of differential characteristics of embryonic organs occurs in the first trimester of pregnancy for both species. Thus, using the combination of the crown‐rump length, macroscopic analysis and optical microscopy, it is possible to predict gestational age more precisely in animals that lack a defined breed and establish an embryonic pattern.
- Relationships among Puberty, Muscle and Fat, and Liveweight Gain during
Mating in Young Female Sheep
- Authors: CA Rosales Nieto; MB Ferguson, H Thompson, JR Briegel, CA Macleay, GB Martin, AN Thompson
Abstract: Greater depths of muscle are associated with better reproductive performance in ewe lambs, but, in adult ewes, reproductive performance also seems to vary with liveweight gain during the mating period. Therefore, in a large field study with Merino ewe lambs, we tested whether the relationships among eye muscle depth (EMD), fat depth (FAT) and reproductive performance depend on liveweight gain during the mating period. We selected lambs with a wide range in phenotypic values for depths of eye muscle (EMD) and fat (FAT) and assigned them to dietary treatments designed to achieve low (LOW, n = 244) or high (HIGH, n = 237) rates of liveweight gain during a 28‐day mating period. The LOW treatment maintained live weight, whereas the HIGH treatment gained 179 ± 3.8 g/day (p
- Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and
- Authors: BC Schimming; PFF Pinheiro, R Matteis, CM Machado, RF Domeniconi
Abstract: Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak‐to‐moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.
- Effect of Conjugated Linoleic Acid on Boar Semen Quality After
Long‐term Refrigeration at 17°C
- Authors: SMP Teixeira; A Chaveiro, F Moreira da Silva
Abstract: In this study, the effect of conjugated linoleic acid (10 trans, 12 cis) (CLA) on refrigerated boar sperm quality parameters up to 14 days at 17°C was assessed. Semen was extended in Androhep and divided into four treatments supplemented with CLA (25, 50, 100 and 200 μm) and control group, then kept for 2 h at 22°C. Afterwards an aliquot of each treatment was removed, and mitochondrial activity, viability, lipid membrane peroxidation (LPO) and stability of the sperm plasma membrane were assessed by flow cytometry. The remaining extended semen was maintained at 17°C until 336 h, repeating the same analysis every 48 h. Regarding percentage of live spermatozoa, no statistical differences were observed among treatments up to 96 h. After this time, viability decreased significantly (p
- Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic
Death Rates in Mares
- Authors: E Malschitzky; AM Pimentel, P Garbade, MIM Jobim, RM Gregory, RC Mattos
Abstract: The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well‐being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well‐being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.
- Rotation of Boar Semen Doses During Storage Affects Sperm Quality
- Authors: M Schulze; K Rüdiger, D Waberski
Abstract: It is common practice to rotate boar semen doses during storage for prevention of sperm sedimentation. In this study, the effect of rotation of boar semen doses during storage on sperm quality was investigated. Manual turning twice daily and automatic rotation five times per hour resulted in the following effects: alkalinization of the BTS‐extender, loss of membrane integrity at day 3, and loss of motility and changes in sperm kinematics during a thermoresistance test at day 5. Using a pH‐stabilized variant of BTS extender, sperm motility and velocity decreased in continuously rotated samples, whereas membrane integrity and mitochondrial activity remain unaffected. It is concluded that rotation of semen samples adversely affects sperm quality and, therefore, should no longer be recommended for AI practice.
- Colour Doppler Ultrasonography as a Tool to Assess Luteal Function in
Santa Inês Ewes
- Authors: LM Figueira; JF Fonseca, EKN Arashiro, JMG Souza‐Fabjan, ACS Ribeiro, E Oba, JHM Viana, FZ Brandão
Abstract: The aim of this study was to evaluate luteal dynamics in the Santa Inês ewes using colour Doppler (CD) ultrasonography. Oestrus was synchronized in nulliparous females (n = 18), and subsequently, they were only teased (n = 6) or teased and mated (n = 12). Blood samples were collected daily for plasma progesterone (P4) concentrations. Ultrasonographic images of corpora lutea (CL) in CD mode were obtained for further analysis in its largest diameter. The CD mode allowed an early sequential monitoring of CL that was visualized by the first time 0.77 ± 0.62 days after ovulation, with luteal area 29.68 ± 13.21 mm2. During the luteogenesis, a progressive increase was observed, followed by a plateau of luteal area, vascularization area and plasma concentrations of P4 reaching maximum values in D11 (124.0 ± 38.0 mm2, 52.78 ± 24.08 mm2 and 11.23 ± 4.89 ng/ml, respectively). In the luteolysis, the plasma concentrations of P4 decreased sharply, whereas luteal and vascularization area gradually. The vascularization area was positively correlated with plasma concentrations of P4 during the luteogenesis (r = 0.22) and luteolysis (r = 0.48). The luteal dynamics of Santa Inês ewes showed patterns similar to those observed in other sheep breeds studied. The CD ultrasonography has the potential to be used as a tool to assess luteal function in sheep.
- Effect of Sildenafil on Pre‐Eclampsia‐Like Mouse Model Induced
- Authors: C Motta; C Grosso, C Zanuzzi, D Molinero, N Picco, R Bellingeri, F Alustiza, C Barbeito, A Vivas, MC Romanini
Abstract: N(omega)‐nitro‐L‐arginine methyl ester (L‐NAME) decreases the vasodilator effect of nitric oxide (NO) and induces pre‐eclampsia in mouse. Sildenafil inhibits the degradation of nitric oxide and increases vasodilation. This study aimed to determine the effects of sildenafil citrate on angiogenesis and oxidative stress at the maternal foetal interface on pre‐eclampsia‐like mouse model induced by L‐NAME. Twenty pregnant mice were divided into four groups: (i) vehicle control; (ii) L‐NAME; (iii) sildenafil; (4) L‐NAME+sildenafil. L‐NAME was administered from day 7 of pregnancy and sildenafil from day 8 until day 16; animals were euthanized on day 17. Placental and foetal sizes and weights were measured; lipid peroxide levels and catalase activity in placental homogenates were determined, and placental vascular endothelia were identified by lectin‐histochemistry using BSA‐I lectin. Western blot analysis was used to determine VEGF expression in placental homogenates. No changes were seen in placental and foetal development in mice with normal pregnancies treated with sildenafil. Treatments with L‐NAME reduced significantly the placental weight and average height and decreased the percentage of the endothelial surface. These alterations may be mediated by the reduction of NO levels in trophoblastic cells, due to the inhibitory effect of L‐NAME on nitric oxide synthase (NOS) synthesis. This effect was offset by the treatment with sildenafil, with an increase in the percentage of the endothelial surface. In conclusion, our results indicate that treatment with sildenafil on pre‐eclampsia mouse model can be used without adverse effects on the concept and its use in the treatment of pre‐eclampsia is promising.
- The Effect of Sperm Concentration and Storage Vessel on
Quercetin‐Supplemented Rabbit Semen During Chilled Storage
- Authors: D Johinke; SP Graaf, R Bathgate
Abstract: Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris‐based extender supplemented with 100 μm quercetin to a concentration of 15, 30 or 60 × 106 spermatozoa/ml, packaged into plastic tubes or 0.5‐ml straws and stored at 15°C. Sperm quality was assessed by computer‐assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 106 spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p
- Is the Production of Embryos in Small‐Scale Farming an Economically
- Authors: Z Sánchez; MA Lammoglia, MA Alarcón, JJ Romero, CS Galina
Abstract: The present assay attempts to evaluate the feasibility of using embryo transfer in small community farmers by in vivo study and by modelling the results obtained. From the total of 59 donor cows, 62.7% responded to treatment, with a significant difference (p = 0.002) in the percentage of the response between breeds, being 90.5% (19/21) in Holstein and 47.4% (18/38) in Brahman. A total of 283 embryos were graded as transferable, while 141 as non‐transferable, without difference in the percentage of transferable embryo by breed (p = 0.18). The mean of transferable embryos graded as class I and II was not different between Holstein and Brahman (p = 0.96 and p = 0.92, respectively); besides, no differences were observed in the other grades (non‐transferable). The highest difference in costs, regardless of its quality by breed, was seen in the lower levels of probable fertility of the embryo transferred, even reaching several hundred dollars. When modelling the expected costs for embryo produced and transferred, values can reach nearly $2000.00 when the probable fertility is only 10%. However, when the probable fertility was 60%, embryo cost was close to $300.00. This technology seems to be viable on average or high‐scale systems, having a superovulatory response between 60 and 80% with 4–6 transferrable embryos. Yet, in small‐scale farming, due to the reduced number of donors and/or recipients, the costs surpass the economical feasibility of the technique.
- Influence of Macrophages on the Rooster Spermatozoa Quality
- Authors: L Kuzelova; J Vasicek, P Chrenek
Abstract: The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low‐density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer‐assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2–3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10–15% of macrophages. Males from macrophage group had lower (p
- Issue Information
- PubDate: 2015-04-23T04:46:35.186347-05:
- Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of
Cooling Curves and Centrifugation Regimes
- Authors: HH Shiomi; RO Pinho, DMA Lima, JB Siqueira, MCR Santos, EV Costa, PS Lopes, SEF Guimarães, JD Guimarães
Abstract: This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800 g for 10 min and 2400 g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400 g for 3 min and freezing 2; M10 – centrifugation at 800 g for 10 min and freezing 2; R3 – centrifugation at 2400 g for 3 min and freezing 1; and R10 – centrifugation at 800 g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.
- Activation of the Albino Sterlet Acipenser ruthenus Eggs by
UV‐Irradiated Bester Hybrid Spermatozoa to Provide Gynogenetic
- Authors: D Fopp‐Bayat; K Ocalewicz
Abstract: Meiotic gynogenesis was induced in the albino form of sterlet Acipenser ruthenus by activation of eggs with UV‐irradiated bester (Huso huso x Acipenser ruthenus) spermatozoa followed by inhibition of the second meiotic division performed by a heat shock. Obtained putative gynogenetic progeny were all albinos. The genetic verification based on three microsatellite DNA markers confirmed the only maternal inheritance of the progeny from the gynogenetic experimental groups. Cytogenetic analysis proved the gynogenetic sterlets were diploids. Application of the albino phenotype together with the molecular and the cytogenetic diagnostic approaches enabled to evaluate the efficiency of the spermatozoa irradiation and application of the heat shock to restore diploid state in the gynogenetic zygotes.
- Monitoring the Foetal Phase of Gestation in the Queen With a
12.5‐MHz Ultrasound Probe and Prediction of the Parturition by
Combining the Measurements of Head and Abdominal Diameters
- Authors: E Topie; D Bencharif, L Briand, D Tainturier
Abstract: Ten gestations in six domestic shorthair cats (Europeans) were monitored daily during the foetal phase of gestation, from the 28th day after the first mating until parturition, using ultrasound with a 12.5‐MHz probe. The development of the various organs over this period was recorded. The diameters of the head (HD) and abdomen (AD) were measured. Skeletal calcification visible on ultrasound occurred in a defined order between the 34th and 40th day of gestation. During the last 30 days of gestation, there was a significant correlation between HD and days before parturition (DBP) (r2 = 0.99) and between AD and DBP (r2 = 0.98). The following equations were obtained: DBP = −2.10*HD (mm) + 50.74; DBP = −1.01*AD (mm) + 42.19. The confidence intervals were stable over the last 30 days of gestation. For the HD, the confidence interval was ±1 day in 53% of cases and ±2 days in 85% of cases. For the AD, the confidence interval was ±1 day in 45% of cases and ±2 days in 77% of cases. A table obtained by combining the HD and AD measurements made it possible to estimate the date of parturition within 2 days with a reliability of over 85%.
- Bovine Circadian Locomotor Output Cycles Kaput (CLOCK) and Clusterin (CLU)
mRNA Quantitation in Ejaculated Crossbred Bull Spermatozoa
- Authors: S Kumar; R Deb, U Singh, I Ganguly, DK Mandal, S Tyagi, M Kumar, G Sengar, S Sharma, R Singh, R Singh
Abstract: Mammalian circadian locomotor output cycles kaput (CLOCK) gene encodes a transcription factor that affects both the persistence and the period of circadian rhythms. Earlier reports suggested that CLOCK gene might be associated with male infertility in human. Present investigation, for the first time, reports that CLOCK gene expresses differentially between good and poor quality crossbred bull semen. The relative expression of CLOCK was significantly (p
- A Pilot Study to Compare Oxidative Status between Organically and
Conventionally Managed Dairy Cattle During the Transition Period
- Authors: A Abuelo; J Hernández, JL Benedito, C Castillo
Abstract: The aim of this study was to assess the redox balance of organically managed dairy cattle (OMC; n = 40) during the transition period and to compare this with conventionally managed cattle (CMC; n = 22). Serum samples of dairy cows from two organic and one conventional farm were taken. Markers of oxidants production [reactive oxygen species] and total serum antioxidant capacity were measured in four different production stages: (i) far‐off dry (2 to 1 months before calving; 44 samples in CMC and 48 in OMC); (ii) close‐up dry (1 month until 3 days before calving; 44 CMC; 54 OMC); (iii) fresh (3 days to +1 month after calving; 44 CMC; 49 OMC); and (iv) peak of lactation (+1 to +3 months; 71 CMC; 78 OMC). Values were compared between production stages and against a metabolic baseline status (4th–5th month of pregnancy; 40 CMC; 30 OMC). Our results indicated that throughout the periparturient period, OMC had lower concentrations of reactive oxygen species, but also a lower antioxidant capacity than CMC. Indeed, when the two components of the redox balance were assessed together through the Oxidative Stress index, the values of this parameter were higher for OMC than for CMC, thereby implying a higher risk of oxidative stress. Therefore, further larger studies are needed to confirm the current observations, as organically reared animals might be exposed to a lack of antioxidants supply.
- Effect of Initial GnRH and Duration of Progesterone Insert Treatment on
the Fertility of Lactating Dairy Cows
- Authors: MG Colazo; DJ Ambrose
Abstract: The study compared response to prostaglandin F2α (PG), synchrony of ovulation and pregnancy per AI (P/AI) in a 5‐ vs a 7‐day Ovsynch + PRID protocol and investigated whether the initial GnRH affects P/AI in lactating dairy cows. Two hundred and seventy‐six cows (500 inseminations) were assigned to one of four timed‐AI (TAI) protocols: (i) PRID‐7G; 100 μg GnRH im, and a progesterone‐releasing intravaginal device (PRID) for 7 days. At PRID removal, PG (500 μg of cloprostenol) was given im. Cows received the second GnRH treatment at 60 h after PRID removal and TAI 12 h later. (ii) PRID‐5G; as PRID‐7G except the duration of PRID, treatment was 5 days and PG was given twice (12 h apart). (iii) PRID‐7NoG; as PRID‐7G except the initial GnRH, treatment was omitted. (iv) PRID‐5NoG; as PRID‐7NoG except the duration of PRID, treatment was 5 days. Response to treatments and pregnancy status at 32 and 60 days after TAI was determined by ultrasonography. The percentage of cows ovulating before TAI was greatest in PRID‐7G (17.1%), and the percentage of cows that did not have luteal regression was greatest in PRID‐5G (9.5%). The overall P/AI at 32 and 60 days did not differ among TAI protocols. However, during resynchronization, cows subjected to the 5‐day protocols had greater (p
- Prostasomes of Canine Seminal Plasma – Zinc‐Binding Ability
and Effects on Motility Characteristics and Plasma Membrane Integrity of
- Authors: M Mogielnicka‐Brzozowska; R Strzeżek, K Wasilewska, W Kordan
Abstract: Prostasomes are small lipid membrane‐confined vesicles that are involved in various fertilization‐related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross‐bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc‐affinity chromatography on a Chelating Sepharose Fast Flow – Zn2 + showed that from 93 to 100% of the prostasome proteins bind zinc ions (P+Zn). SDS‐PAGE revealed that canine P+Zn comprised four protein bands, with low molecular weights (10.2–12 kDa). We have also shown a positive effect of prostasomes (p
- Differences Between High‐ and Low‐Motility Buffalo Sperm
Identified by Comparative Proteomics
- Authors: Y‐L Huang; Q Fu, L Yang, J‐L Guan, H Pan, F‐M Chen, K‐L Lu, M Zhang
Abstract: This study was undertaken to investigate differences in protein expression between high‐ and low‐motility sperm of swamp buffalo. The research used two‐dimensional gel electrophoresis (2DE) coupled to matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐TOF/TOF‐MS) to analyse the different proteins. The results showed 18 different expression protein spots between high‐ and low‐motility buffalo sperm; eight of these proteins were up‐regulated in low‐motility sperm, five were down‐regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl‐CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low‐motility sperm and provide clues for finding molecular markers associated with sperm motility.
- Generation of Foxo3‐targeted Mice by Injection of mRNAs Encoding
Transcription Activator‐like Effector Nucleases (TALENs) into
- Authors: P Zhu; Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang, D Shi
Abstract: In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3‐targeted mice were generated by injection of mRNAs encoding transcription activator‐like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio‐companies. The TALENs mutagenic non‐homologous end‐joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK‐293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN‐mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN‐mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3‐independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3−/+), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide‐type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs‐based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.
- Transabdominal Ultrasonography for Pregnancy Diagnosis and Estimation of
Gestational Age in Dromedary Camels
- Authors: A Ali; R Derar, F Al‐Sobayil
Abstract: The objective of this study was to evaluate the use of transabdominal ultrasonography as a method for pregnancy diagnosis and establish some foetal biometrics guidelines of pregnancy staging. Transabdominal ultrasonography was performed weekly on six pregnant camels from the 4th to the 19th week and at 2‐week intervals from the 20th to the 52nd week of gestation. Six areas were selected for examination: caudal abdominal (CAA), inside the thigh, above the base of the udder; middle abdominal (MIA), from the base of the udder to the umbilicus; and cranial abdominal (CRA), from the umbilicus to the xiphoid cartilage. Several foetal parameters including orbital diameter (ORD), biparietal diameter (BPD), abdominal diameter (ABD), chest depth (CHD) and ruminal diameter (RUD) were measured using standardized scan plans. The relationships between the gestational age and the foetal parameters were evaluated. From the 6th to the 12th week, the conceptus was always observed through the left CAA approach. Between the 13th and 27th week, the foetus was entirely visualized by the MIA examination. From the 28th to the 52nd week, the conceptus was mainly detected through the left CRA approach. The foetal parameters were measured frequently during mid‐ and late gestation. All regression and correlation coefficients were highly significant (p
- Interactions Among Different Devices and Electrical Stimulus on the
Electroejaculation of Captive Agoutis (Dasyprocta leporina)
- Authors: TS Castelo; ALP Souza, GL Lima, GCX Peixoto, LB Campos, MF Oliveira, AR Silva
Abstract: The interactions among different electroejaculation devices associated with serial or continuous stimuli were investigated to improve the efficiency of the electroejaculation for semen collection in agoutis. Ten sexually matured male Dasyprocta leporina were restrained by the intramuscular administration of xylazine–ketamine association. Each individual was randomly subjected to four electroejaculation protocols, by combining two devices (one presenting longitudinal electrodes emitting square waves and other presenting ring electrodes emitting sine waves) and two electrical stimuli protocols (serial or continuous). A total of 40 attempts for electroejaculation were conducted in agoutis, being 10 per treatment. The most efficient treatment in providing ejaculates containing sperm (p
- The Usefulness of Captive Kept Capercaillie (Tetrao urogallus L.) as the
Semen Donors for Artificial Insemination and Gene Pool Preservation In
- Authors: ET Łukaszewicz; AM Kowalczyk
Abstract: Captive breeding of birds threatened by extinction in zoological gardens or other closed aviary centres is one of the methods allowing their protection and gene pool preservation ex situ in vivo. Such birds are usually kept in captivity lifelong and serve as parents of several new generations that can be further released into natural environment, or males are used as semen donors for artificial insemination and gene banking. Therefore, the fecundity of such flocks (number of laid egg and spermatozoa quantity and quality) is very important. The aim of this study was to evaluate the usefulness of captive kept capercaillie (Tetrao urogallus L.) as semen donors in three subsequent reproductive seasons, based on the assessment of manually collected semen quality. Male response to dorso‐abdominal massage, ejaculate volume, sperm concentration, motility and morphology were evaluated individually at three succeeding years. Depending on individual male properties and year of collection, the number of positive reactions to semen collection attempts (i.e. ending with ejaculation) varied from 44.4% to 100.0%; single ejaculate volume ranged from 10 to 300 μl, spermatozoa concentration from 10 × 106 per ml to 3520 × 106 per ml and percentage of live morphologically normal spermatozoa from 19.3 to 80.3%. The highest average value (66.7) of semen quality factor (SQF) was noted for a 2‐year‐old male (varying from 1.9 to 258.1), while the lowest for ten‐ (4.8; varying from 0.1 to 17.0) and 7‐year‐old (6.6; varying between 0.6 and 13.6). Assuming that for AI purposes, the ejaculate quality has to be at minimum 10 SQF, obtained results indicate that majority of capercaillie kept in captivity, both young (2–3 years old) and older (up to 10 years old), can be valuable semen producers in succeeding seasons.
- Localization of CD9 Molecule on Bull Spermatozoa: Its Involvement in the
- Authors: J Antalíková; J Jankovičová, M Simon, P Cupperová, K Michalková, Ľ Horovská
Abstract: Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA‐50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen‐thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti‐CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.
- Reproductive Performance and Reconception of Nellore Cows According to
Their Pure‐ or Cross‐Bred Calves
- Authors: WB Rodrigues; FS Wechsler, E Nogueira
Abstract: The objective of the present research was to evaluate the effect of a calf's genetic group on the productive and reproductive efficiency of its Nellore dam. Fixed‐time artificial insemination was applied to 800 cows using semen extracted from Nellore, Simmental and Angus Red bulls. Four hundred eleven cows calved, producing 119 Nellore, 103 ½Simmental–½Nellore and 189 ½Nellore–½Angus Red calves. The second mating period, which paired Nellore cows with Simmental bulls, was initiated 10 days after parturitions began and lasted for 5 months. Based on the two successive parturitions, the cumulative parturition rate for calving periods of 3, 4 and 5 months was calculated. Although no significant difference was observed for birth weight among the genetic groups, cross‐bred calves weighed, on average, 10% more than did pure‐bred calves at the age of 205 days. Nellore dams experienced a gestation period that was 7 days longer than did the cross‐bred dams, and the former showed a higher parturition rate at 90 and 120 days of the calving season, but not at 150 days (calving rates of 80.6, 76.4 and 76.2% for mothers of Nellore, ½Nellore–½Angus Red and ½Nellore–½Simmental, respectively, p > 0.05). At 90 and 120 days, Nellore dams produced more kg of calf per mated dam. In conclusion, in a short breeding season, Nellore dams nursing pure‐bred Nellore calves were found to have a higher biological efficiency compared with Nellore dams nursing cross‐bred calves.
- Expression of Mitochondria‐Associated Genes (PPARGC1A, NRF‐1,
BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries
- Authors: G Zhang; Y Wan, Y Zhang, S Lan, R Jia, Z Wang, Y Fan, F Wang
Abstract: Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.
- Expression of Heparin‐Binding EGF‐Like Growth Factor
(HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and
Interferon‐τ on Prostaglandin Production
- Authors: K Takatsu; TJ Acosta
Abstract: Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p
- Malignant Seminoma in Two Unilaterally Cryptorchid Stallions
- Authors: V De Lange; K Chiers, L Lefère, M Cools, C Ververs, J Govaere
Abstract: Two unilateral cryptorchid stallions were referred to the clinic because of chronic debilitating condition with emaciation. Rectal examination, and ultrasound and gross examination revealed in both animals an abdominal mass, caudally of the kidney, and multiple nodules spread over the abdomen. Histologic analysis revealed an intra‐abdominal malignant seminoma with intraperitoneal and renal metastasis. Interestingly, a seminoma was also present in the descended testis of the draught horse.
- P53‐Mediated Repression of the Reprogramming in Cloned Bovine
Embryos Through Direct Interaction with HDAC1 and Indirect Interaction
- Authors: PJ Ma; H Zhang, R Li, YS Wang, Y Zhang, S Hua
Abstract: P53 is a transcriptional activator, regulating growth arrest, DNA repair and apoptosis. We found that the expression level of P53 and the epigenetic profiles were significantly different in bovine somatic cell nuclear transfer embryos from those in vitro fertilization (IVF) embryos. So we inferred that abnormally expression of P53 might contribute to the incomplete reprogramming. Using bovine foetal fibroblasts, we constructed and screened a highly efficient shRNA vector targeting bovine P53 gene and then reconstituted somatic cell nuclear transfer embryos (RNAi‐SCNT). The results indicated that expression levels of P53 were downregulated significantly in RNAi‐SCNT embryos, and the blastulation rate and the total number of cell increased significantly. Moreover, methylation levels of CpG islands located 5′ region of OCT4, NANOG, H19 and IGF2R in RNAi ‐SCNT embryos were significantly normalized to that IVF embryos, and the methylation levels of genome DNA, H3K9 and H4K5 acetylation levels were also returned to levels similar to the IVF embryos. Differentially expressed genes were identified by microarray, and 28 transcripts were found to be significantly different (> twofolds) in RNAi‐SCNT embryos compared to the control nuclear transfer embryos (SCNT). Among the 28 differentially expressed transcripts, just HDAC1 and DNMT3A were closely associated with the epigenetic modifications. Finally, ChIP further showed that P53 might repress the epigenetic reprogramming by regulating HDAC1 directly and DNMT3A indirectly. These findings offer significant references to further elucidate the mechanism of epigenetic reprogramming in SCNT embryos.
- Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep
- Authors: LF Crocomo; WC Marques Filho, CMV Ulian, NS Branchini, DT Silva, CL Ackermann, FC Landim‐Alvarenga, SD Bicudo
Abstract: Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (−) at 38.5°C and 5% CO2. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/−) exhibited total expansion while in both Rosco groups (+/−) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p
- Assessment of Genetic Variability in the Coding Sequence of Melatonin
Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India
- Authors: VK Saxena; BK Jha, AS Meena, HK Narula, D Kumar, SMK Naqvi
Abstract: Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane‐bound G‐coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out‐of‐season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen‐2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out‐of‐season breeding. GG and CC genotypes were having a higher prevalence in the studied population.
- Aquagrams of Raw Milk for Oestrus Detection in Dairy Cows
- Authors: G Takemura; G Bázár, K Ikuta, E Yamaguchi, S Ishikawa, A Furukawa, Y Kubota, Z Kovács, R Tsenkova
Abstract: The purpose of this research was to develop rapid and cost‐effective method for oestrus detection in dairy cows by means of near infrared spectroscopy and aquaphotomics, using raw milk from individual cows. We found that aquaphotomics approach showed consistent specific water spectral pattern of milk at the oestrus periods of the investigated Holstein cows. Characteristic changes were detected especially in foremilk collected at morning milking. They were reflected in calculated aquagrams of milk spectra where distinctive spectral pattern of oestrus showed increased light absorbance of strongly hydrogen‐bonded water. Results showed that monitoring of raw milk near infrared spectra provides an opportunity for analysing hormone levels indirectly, through the changes of water spectral pattern caused by complex physiological changes related to fertile periods.
- Chlamydia abortus in Cows Oviducts, Occasional Event or Causal
- Authors: S Appino; L Vincenti, A Rota, S Pellegrini, MN Chieppa, V Cadoni, P Pregel
Abstract: Fifty‐seven genital tracts of regularly slaughtered culled Piedmontese cows, aged 7.4 ± 4.3 years (mean ± SD), range: 2.6–15.6 years, were grossly and microscopically examined. DNA extracted from oviducts was subjected to PCR to evaluate the presence of Chlamydia spp. The 15 PCR‐positive oviducts were subjected to Sanger sequencing and showed the presence of Chamydia abortus, with an identity range between 99 and 100%. Nine of the PCR‐positive samples belonged to the 24 animals with a normal macroscopic appearance of the whole genital tract (percentage of positive oviducts in normal genital tracts 9/24 = 37.5%), while six belonged to the 33 genital tracts with lesions in one or more organs (percentage of positive oviducts in pathological genital tracts 6/33 = 18.1%); of these, a single animal had salpingitis. The detection of C. abortus in bovine oviducts is of particular interest because it has never been previously investigated or reported.
- The Concentrations of Inflammatory Cytokines and Acute‐Phase
Proteins in the Peripheral Blood and Uterine Washings in Cows with
- Authors: P Brodzki; K Kostro, A Brodzki, J Ziętek
Abstract: The development of pyometra in cows depends largely on the state of local immunity of the uterus. The objective of the study was to evaluate the concentration of the following proinflammatory cytokines: tumour necrosis factor (TNF‐α) and interleukin‐6 (IL‐6); anti‐inflammatory cytokine: interleukin‐10 (IL‐10); and acute‐phase proteins (APPs): haptoglobin (Hp) and serum amyloid A (SAA), in serum and uterine washings in cows with pyometra and healthy animals. The study was performed on 20 cows divided into two groups based on the results of cytological and ultrasonographic tests: a pyometra and a healthy group (10 cows per group). Experimental material consisted of blood serum and uterine washings. The levels of the following cytokines, TNF‐α, IL‐6, IL‐10 and APPs – Hp and SAA, in the study material were determined by ELISA. The results showed that the values of TNF‐α, IL‐6, IL‐10 as well as SAA and Hp were significantly higher in serum of cows with pyometra compared to controls (p
- Effects of Ovariohysterectomy on Oxidative Stress Markers in Female Dogs
- Authors: M Szczubial; M Kankofer, M Bochniarz, R Dąbrowski
Abstract: Numerous studies reported an increase of oxidative stress increases in both women and female laboratory animals after ovariectomy. However, there is little information about the evaluation of antioxidative/oxidative status in ovariectomized dogs. The purpose of this study was to examine the changes in oxidative stress markers after ovariohysterectomy (OHE) in female dogs. The study included eighteen healthy mongrel female dogs. Blood samples were collected immediately before surgery and 14 and 30 days after surgery. Following parameters of oxidative stress intensity were determined: the erythrocyte activity of glutathione peroxidase (GSH‐Px) and superoxide dismutase (SOD) as well as the plasma concentrations of thiobarbituric acid reactive substances (TBARS), radical cations of N,N, diethylpara‐phenylene diamine (RC‐DEPPD), sulfhydryl groups (SH groups), bityrosine and formylkynurenine. The activity of GSH‐Px increased markedly, although not significantly, 14 days after OHE and then significantly decreased at 30 days after OHE. A significant increase in plasma TBARS, bityrosine and formylkynurenine concentrations and a decrease in SH group content were concurrently noted at 30 days after surgery. Acquired results suggested that a loss of control over ROS production occurred in female dogs after OHE, which could lead to oxidative stress in the late post‐operative period. In conclusion, our findings indicated that OHE is related with the risk of oxidative stress in the late period after operations. Given that oxidative stress contributes to the pathogenesis of various diseases, this may suggest an increased risk of disorders in ovariectomized female dogs; however, further studies are necessary to confirm this hypothesis.
- Comparative Expression Analysis of Gametogenesis‐Associated Genes in
Foetal and Adult Bubaline (Bubalus bubalis) Ovaries and Testes
- Authors: SM Shah; N Saini, S Ashraf, M Zandi, MK Singh, RS Manik, SK Singla, P Palta, MS Chauhan
Abstract: This study was conducted to identify and analyse the expression of gametogenesis‐associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis‐associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p
- Stem Cells from Foetal Adnexa and Fluid in Domestic Animals: An Update on
Their Features and Clinical Application
- Authors: E Iacono; B Rossi, B Merlo
Abstract: Over the past decade, stem cell research has emerged as an area of major interest for its potential in regenerative medicine applications. This is in constant need of new cell sources to conceive regenerative medicine approaches for diseases that are still without therapy. Scientists drew the attention towards alternative sources such as foetal adnexa and fluid, as these sources possess many advantages: first of all, cells can be extracted from discarded foetal material and it is non‐invasive and inexpensive for the patient; secondly, abundant stem cells can be obtained; and finally, these stem cell sources are free from ethical considerations. Cells derived from foetal adnexa and fluid preserve some of the characteristics of the primitive embryonic layers from which they originate. Many studies have demonstrated the differentiation potential in vitro and in vivo towards mesenchymal and non‐mesenchymal cell types; in addition, the immune‐modulatory properties make these cells a good candidate for allo‐ and xenotransplantation. Naturally occurring diseases in domestic animals can be more ideal as disease model of human genetic and acquired diseases and could help to define the potential therapeutic use efficiency and safety of stem cells therapies. This review offers an update on the state of the art of characterization of domestic animals' MSCs derived from foetal adnexa and fluid and on the latest findings in pre‐clinical or clinical setting of the stem cell populations isolated from these sources.
- Effect of Seminal Plasma on Post‐Thaw Quality and Functionality of
- Authors: A Ledesma; J Manes, G Ríos, J Aller, A Cesari, R Alberio, F Hozbor
Abstract: We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post‐thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.
- Congenital Abnormality of the Vagina Complicated by
Haemato‐Pyocolpos in a 1‐Year Labrador Retriever
- Authors: S Alonge; S Romussi, V Grieco, GC Luvoni
Abstract: A 1‐year‐old female Labrador retriever was referred with a few days history of haematic‐like vulvar discharge. Physical examination, vaginal inspection and palpation did not reveal any remarkable finding. Transabdominal ultrasound showed echogenic fluid accumulation in the vagina suggesting haemato‐pyocolpos. An exploratory laparotomy was performed: a well‐delimited ectasic vagina was identified. Ovariohysterectomy and partial vaginectomy and vaginoplasty were performed to spay the bitch and to remove the ectasic vagina. Post‐operative recovery and 12‐month follow‐up were uneventful. Clinical, morphological and histological findings were consistent with a congenital abnormality of the muscular layer of the vagina complicated by haemato‐pyocolpos. The disorganization of the vaginal tunica muscularis may have acted as locus minoris resistentiae in the vaginal wall. The organ was dilated and atonic due to the gradual accumulation of physiological fluids complicated by an overgrowth of genital bacteria. This congenital disorder has to be taken into account as differential diagnosis of haemato‐pyocolpos with vaginal discharge in young bitches.
- Determination of Appropriate Cryopreservation Protocols for Epididymal Cat
- Authors: K Buranaamnuay
Abstract: Effects of Equex and glycerol additions and sample dilution step on frozen–thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one‐ (groups III and IV) or two‐ (groups I, II, V and VI) step dilution. For one‐step dilution, the pellets were resuspended in plain egg yolk‐Tris medium (EYT) + 5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4°C, 1 h). For two‐step dilution, the pellets were resuspended in EYT (I and V) and in EYT + 3% glycerol (II and VI), cooled and further diluted with EYT + 10% glycerol with (I)/without (V) 1% Equex and with EYT + 7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze–thawing, no differences (p > 0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p = 0.005 to p = 0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p > 0.05) and was higher than the rest (p 0.05) except the lower percentages of viability in III (p = 0.008 to p = 0.3) and of HOST in IV (p = 0.005 to p = 0.2). Two‐step dilutions with Equex (I, II) were more beneficial for the FITC at 2 h than without Equex (V) (p = 0.005 and p = 0.02) and than one‐step dilutions (III, IV) (p
- Neuroendocrine, Metabolic and Genomic Cues Signalling the Onset of Puberty
- Authors: CA Meza‐Herrera; A Gonzalez‐Bulnes, RT Kridli, M Mellado, CF Arechiga‐Flores, H Salinas, JM Luginbuhl
Abstract: Puberty is the result of a dynamic interaction between genetic factors and environmental cues, all of which lead to the attainment of reproductive capacity. Thus, significant changes in hormone secretion occur from the pre‐pubertal to the pubertal stage. The objective of this review is to provide an update of some endocrine, physiological, metabolic and genetic concepts involved in the establishment of the hypothalamic‐hypophyseal‐gonadal axis function promoting the onset of the reproductive function during puberty. To achieve this purpose, basic aspects of the function of the hypothalamic‐hypophyseal‐gonadal axis, the control of the axis by neurotransmitters and the interaction between reproductive function and metabolic status will be considered. Finally, the role of the novel kisspeptin system and the GPR54 receptor as modulators of puberty will be considered, in addition to the hierarchical expression of the main genes acting as regulators of the onset of puberty.
- Comparison of Two Different Programmes of Ovulation Synchronization in the
Treatment of Ovarian Cysts in Dairy Cows
- Authors: N Gundling; S Drews, M Hoedemaker
Abstract: It was the aim of this field study to evaluate two different protocols of ovulation synchronization for the treatment of ovarian cysts and their effect on reproductive performance in dairy cows. In addition, factors with a possible influence on treatment success and pregnancy outcome as well as costs per pregnancy were analysed. The study was performed with 130 German Holsteins with ovarian cysts diagnosed on days 55 to 60 postpartum. Cows belonging to group 1 (n = 65) received a modified ovsynch protocol [day 0: 0.15 mg cloprostenol (PGF) + 0.02 mg buserelin acetate (GnRH); day 14: PGF; day 16: GnRH]. Group 2 (n = 65) was treated with the conventional ovsynch protocol (day 0: GnRH; day 7: PGF; day 9: GnRH). Timed artificial insemination was performed 20 to 24 h later. Cows without ovarian cysts served as controls. Treatment success (disappearance of the ovarian cyst) after the first ovsynch cycle was higher in group 1 (66.2%) than in group 2 (23.1%, p
- Luteal Characteristics and Progesterone Production on Day 5 of the Bovine
- Authors: MP Green; GE Mann, MG Hunter
Abstract: In this study we have examined luteal function in non‐lactating and late lactation dairy cows on day 5 of the cycle, during the period of the post‐ovulatory progesterone rise. Comparison of luteal progesterone content and in vitro synthetic capacity with circulating plasma progesterone demonstrated that circulating progesterone concentration is a function of total luteal activity rather than the activity of individual units of tissue. Incubation of luteal tissue in vitro demonstrated stimulatory activity of LH and IGF‐I, and to a greater degree IGF‐II, on luteal progesterone synthesis. Finally the study showed no effect of double ovulation on luteal function. Occurrence of double ovulation in 35% of animals was not associated with any difference in luteal function or plasma progesterone concentrations.
- Effect of Time for Mating and Gestation Length on Reproductive Efficiency
- Authors: Y Shimatsu; H Yuzawa, K Aruga, M. Nakura
Abstract: The purpose of this study was to examine the effect of time for mating and gestation length on reproductive efficiency in dogs. Groups of eight, six and six beagle bitches were mated with a total of three sires on days 3, 5 and 7, respectively, after the luteinizing hormone (LH) surge. All the bitches whelped successfully. The gestation lengths (the intervals from the LH surge to the whelping) were 65.1 ± 1.9, 65.5 ± 1.9 and 68.0 ± 1.8 days, respectively. This length of mating 7 days after the LH surge was significantly longer than that of mating 3 and 5 days after the LH surge (p
- Successful Low Dose Insemination of Flow Cytometrically Sorted Ram
Spermatozoa in Sheep
- Authors: SP De Graaf; G Evans, WMC Maxwell, JA Downing, JK O'Brien
Abstract: The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo® SX) and cryopreservation was assessed after low‐dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen‐impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 × 106, 5 × 106 or 15 × 106 motile sorted frozen‐thawed (S1, S5, or S15 respectively) or non‐sorted frozen‐thawed (C1, C5 or C15 respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 × 106 motile non‐sorted frozen‐thawed spermatozoa (C50) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C50 (24/38, 63.2%), C15 (37/54, 68.5%), S15 (38/57, 66.7%), S5 (37/56, 66.1%) and S1 (32/52, 61.5%) groups (p > 0.05), but lower for C5 (19/48, 39.6%) and C1 (19/55, 34.5%) treatments (p
- Bovine Endothelial Cells Interact with Fully‐luteinized, but Not
Luteinizing, Granulosa Cells in the mRNA Expression of Endothelin‐1
System in Response to Prostaglandin F2α
- Authors: K Shirasuna; S Watanabe, D Yamamoto, M Hayashi, K Nagai, A Miyamoto
Abstract: The corpus luteum (CL) undergoes regression by prostaglandin (PG)F2α from uterus and endothelin‐1 (ET‐1) plays an important role during luteolysis as a local mediator of PGF2α in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET‐1 synthesis by PGF2α. We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully‐luteinized granulosa cells (GC) and evaluated the effect of PGF2α on the expression for mRNA of ET‐1 system by using real‐time RT‐PCR. PGF2α stimulated the expression of preproET‐1 and endothelin converting enzyme‐1 mRNA only in the co‐cultures of BAEC with fully‐luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully‐luteinized GC enhance the capability of BAEC to produce ET‐1 in response to PGF2α. This mechanism may contribute to the local induction of luteolytic action of PGF2α which is dependent on the age/maturation of the CL.
- Ovum Pick‐up in Dairy Heifers: Does it Affect Animal
- Authors: S Petyim; R Båge, A Madej, B Larsson
Abstract: The aim of the present study was to assess the effects of the ovum pick‐up (OPU) technique on animal well‐being. Eight dairy heifers were subjected to 4 months of twice‐weekly OPU. The physiological response to OPU was recorded in four heifers at two sessions, at the beginning (time 1) and at the end (time 2) of the 4‐month period. Heart rates were measured and blood was analysed for cortisol, vasopressin and PG‐metabolite before, during (every 5 and 2 min), and after the OPU sessions. Reactions to each subprocedure of OPU (‘restraint’, ‘epidural’, ‘device in’ and ‘puncture’) were closely observed. In all heifers, reactions to the OPU procedures were also noted throughout the experimental period, and changes in routine behaviour, oestrous behaviour, body temperature, or other clinical traits were recorded. Subsequent to the experiment, the ovaries and tails were carefully inspected. At time 1, there was an insignificant increase in heart rate and cortisol throughout the OPU procedure. At time 2, these two parameters increased significantly, but both parameters declined to pre‐OPU levels 10 min after completion of the procedure. No significant changes were seen in vasopressin or PG‐metabolite at time 1 and time 2. Behaviourally, the heifers showed the strongest response to epidural anaesthesia, with a tendency for more intense response during the late 4‐month sessions. The response to ‘device in’ and ‘puncture’ varied among individuals independently of time. There were no changes in the routine or oestrous behaviour throughout the experiment and no signs of clinical disorders. No major pathological changes were macroscopically seen in the ovaries and tails subsequent to the 4 months of OPU. In conclusion, the heifers showed a response to OPU, mostly to administration of epidural anaesthesia. However, we demonstrated that epidural anaesthesia can be administered in a way causing less discomfort.
- Infertility in a Dog due to Proximal Cytoplasmic Droplets in the
Ejaculate: Investigation of the Significance for Sperm Functionality In
- Authors: AI Peña; M Barrio, JJ Becerra, LA Quintela, PG Herradón
Abstract: A 4‐year‐old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 × 106), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida‐binding assay, using canine oocytes derived from frozen‐thawed ovaries, was performed in order to investigate the zona‐binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida‐binding assay, ovaries were thawed and minced in phosphate‐buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35–40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5°C and 5% CO2 in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 μl droplets of CCM were incubated for 1 h. Afterwards, 5 μl of CCM containing 25 000 spermatozoa were added to each droplet and co‐incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.
- Early Detection of Pregnancy and Embryonic Loss in Dairy Cattle by ELISA
- Authors: G Gábor; F Tóth, L Ózsvári, Zs Abonyi‐Tóth, RG Sasser
Abstract: Over a 25‐month period 8118 blood samples were assayed for the presence of the serum pregnancy specific‐protein B (PSPB) and progesteron (P4) concentrations on three Hungarian large‐scale dairy farms. Pregnancy (n = 4085) was checked by BioPRYN assay at 30–36 days post‐insemination (PI). Samples from all cows that tested not pregnant and from cows with an optical density (OD) reading in the BioPRYN test that was between 0% and 30% above the cutoff OD value were tested for serum P4 concentration. According to serum P4 concentration, cows were assigned to three categories: high (>4 ng/ml), medium (2–4 ng/ml) and low (
- Distribution of Spermatozoa and Embryos in the Female Reproductive Tract
after Unilateral Deep Intra Uterine Insemination in the Pig
- Authors: P Tummaruk; P Sumransap, M Techakumphu, A Kunavongkrit
Abstract: The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6–8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 × 106 motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 × 106 motile spermatozoa. The sows were anesthetized and ovario‐hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero‐tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 × 106 motile spermatozoa. The sows were anesthetized 61.1 ± 12 h after insemination (48–72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI‐group, and from unilateral side of the reproductive tract in the DIUI‐group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI‐ than the DIUI‐group (p