- Associations of Leptin and Pituitary‐Specific Transcription Factor
Genes’ Polymorphisms with Reproduction and Production Traits in
- Authors: MAF Nasr; A Awad, IE El Araby
Abstract: This study aimed to detect the genetic variability in Leptin and Pit‐1 genes using polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing also to explore their possible associations with reproductive and productive traits of Egyptian buffaloes. Regarding Leptin gene, three genotypes (AA, AG and GG) were identified with frequency of 0.54, 0.40 and 0.06, respectively, and the genotypes were distributed according to the Hardy–Weinberg equilibrium. Allele A was comparatively higher than G with frequency of 0.74 and 0.26, respectively. For Pit‐1 gene, the association could not be performed due to the monomorphism (BB). The results showed that AA genotypes were found to be superior in most of production and reproduction traits. AA genotypes yielded more milk (2332.34 kg, p = 0.04) with higher fat% (6.10, p = 0.004) and fat yield (155.75 kg, p = 0.06), reach peak milk production at 42.19 days and required 2.19 services for conception in comparison with GG genotypes. Birthweight of animals with AA genotype was lesser than with GG genotype (39.35 and 43.67 kg, p = 0.02, respectively). The days open is numerically better in AA genotype animals (99.35 days), but the difference between the three genotypes was non‐significant. The distinct significant associations reported in this study suggested that Leptin is reputable candidate genetic marker, which might be used to enhance animals’ genetic potential for milk production in conjunction with reproduction.
- Overexpression of C/EBPβ Affects The Cell Cycle Regulators and
Spermatogenesis Related Genes Expression And Function of Bovine Sertoli
- Authors: K Tang; Y Jin, F Chen, L Wang
Abstract: CCAAT/enhancer‐binding protein beta (C/EBPβ), an important transcriptional factor, plays a pivotal role in the regulation of female germ cell development. However, the role of C/EBPβ on the development of male germ cells has not been reported. In this study, we constructed the recombinant adenovirus plasmids of bovine C/EBPβ gene and harvested the subsequent adenoviruses, and then assessed the mRNA levels of spermatogenesis‐related genes (real‐time PCR) and secretion of inhibin B after 48 h of Ad‐C/EBPβ recombinant adenovirus infection in bovine sertoli cells (SCs). We found that overexpression of exogenous C/EBPβ gene upregulated the mRNA expressions of spermatogenesis‐related genes, including Pdgfa, Claudin, Caspase‐3, Occludin, kit1 and Cyclin E, and decreased the mRNA levels of Cyclin D1. Meanwhile, overexpression of exogenous C/EBPβ gene significantly increases the amounts of secreted inhibin B. In conclusion, the results indicate that the C/EBPβ gene plays an important regulatory role in regulation of the cell cycle regulators and spermatogenesis‐related genes expression and function of bovine SCs.
- Lysophosphatidic Acid Synthesis and its Receptors’ Expression in the
Bovine Oviduct During the Oestrous Cycle
- Abstract: Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early‐luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early‐luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus–oocyte complex.
- The Central Effect of β‐Endorphin and Naloxone on The
Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The
Hypothalamic‐Pituitary Unit of Follicular‐Phase Ewes
- Abstract: The effects of prolonged, intermittent infusion of β‐endorphin or naloxone into the third cerebral ventricle of follicular‐phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme‐linked immunoabsorbent assay. Activation or blockade of μ‐opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with β‐endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that β‐endorphinergic system in the hypothalamus of follicular‐phase ewes affects directly or via β‐endorphin‐sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic‐pituitary axis.
- Changes in the Expression of the Prolactin Receptor (PRLR) Gene in
Different Physiological Stages in the Mammary Gland of the Iranian Adani
- Authors: S Morammazi; AA Masoudi, R Vaez Torshizi, A Pakdel
Abstract: The actions of prolactin hormone are mediated by prolactin receptor (PRLR), and proliferation and differentiation of secretory mammary epithelium are dependent on the presence of its receptors. To understand the PRLR expression pattern in mammary gland of dairy goat during different lactation stages, in this study, we first estimated the milk yield breeding value by multitrait random regression model and then compared the expression of the gene in different physiological stage of mammary gland between high‐ and low‐breeding value groups. We assayed the transcription level of the gene by quantitative real‐time PCR method, and its outcomes were analysed by a statistical model containing breeding value groups, sampling times and their interactions as fixed effects. The results indicated that the expression levels of PRLR gene were significantly upregulated in the drying stage (p
- Comparison of DNA Fragmentation Assay in Frozen‐Thawed Cat
- Abstract: DNA fragmentation of frozen‐thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA®), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p
- Equine Chorionic Gonadotropin and Human Chorionic Gonadotropin Stimulation
Increase the Number of Luteinized Follicles and the Progesterone Level
Compared with Cabergoline Stimulation in Anoestrus Bitches
- Abstract: In this study, ovarian morphologies and blood progesterone concentrations following oestrous induction in bitches were examined. Fifty‐three clinically healthy anoestrus bitches received cabergoline at a daily dose of 5 μg/kg of body weight per os for 21 days (group I) or subcutaneous equine chorionic gonadotropin at a dose of 20 IU/kg of body weight for five consecutive days with an additional 500 IU s.c. per bitch of human chorionic gonadotropin on the last day of treatment (group II). Twenty bitches that spontaneously displayed oestrous signs were left untreated and served as controls (group III). The induced oestrous rates and ovulation rates in groups I and II were 60.0% vs 64.3% and 86.7% vs 83.3%, respectively. Morphological assessments of the ovarian structures after ovariohysterectomy revealed an increase in the number of luteinized follicles and cysts in group II compared with the two other groups (p
- Effect of Different Levels of Silymarin and Caproic Acid on Storage of Ram
Semen in Liquid Form
- Abstract: Two experiments were designed to evaluate the effect of silymarin on stored spermatozoa using four rams. In experiment 1, silymarin was evaluated as a supplement for Tris–glucose extender. Semen samples (n = 20) were diluted with extender containing 0, 50, 100, 150 and 200 μg/ml silymarin and incubated at 5°C for 72 h. Membrane integrity, acrosome integrity, sperm viability and motility were evaluated at 72 h. Concentration of malondialdehyde (MDA) was determined after 48 h. Membrane integrity was higher in 100 μg/ml silymarin (65.2%) than control group (43.2%, p
- Detection and isolation of Toxoplasma gondii from fresh semen of naturally
infected dogs in Southern Brazil
- Abstract: The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.
- Comparison of Intrauterine Antibiotics versus Ozone Medical Use in Sheep
with Retained Placenta and Following Obstetric Assistance
- Abstract: This study outlines a new approach to reproductive tract treatment using ozone foam spray for certain ovine obstetrical problems, such as retained foetal membranes and possible uterine infections following obstetric assistance (OA), in comparison with classical antibiotics treatments. The study was conducted on 256 ewes from 11 sheep farms in north‐western Croatia. A total of 139 ewes were diagnosed with dystocia (DT) and 49 with retention of placenta (RP). Ewes with RP were treated either with ozone foam spray (Riger spray G; Novagen®) applied into the body of the uterus for 2–3 s (first or RPO group; n = 24) or with two foaming, intrauterine tablets of oxytetracycline hydrochloride (Geomycin® F) (second or RPA group; n = 25). The third and fourth groups consisted of ewes that received OA for dystocia (including ringwomb, foetal oversize and assistance of abnormal position and posture). The third group (DTO; n = 70) was treated with ozone foam spray, while ewes in fourth group (DTA; n = 69) were treated with antibiotics. The ewes in the control group (CTL) with physiological puerperium were randomly selected (n = 70) from all herds. Transrectal ultrasonography (transversal diameter of uterine horns) was used for the control of uterus regression on days 2 and 25 after parturition. There was a difference in transversal uterine horn diameter in the RP groups, that is RPO and RPA (5.40 ± 0.53 cm vs. 5.43 ± 0.40 cm), ewes with dystocia, that is DTO and DTA (5.37 ± 0.49 cm vs. 5.54 ± 0.60 cm) and ewes from the CTL group (4.98 ± 0.35 cm) one day after parturition. Average transversal uterine diameter of all groups at day 25 post‐partum was 1.80 ± 0.15 cm. The intrauterine ozone treatment in ewes with RP and after manual obstetrics attained similar results to spontaneously delivered ewes (CTL group), showed as the physiological regression of the uterus with a similar transversal diameter without the presence of lochia in the uterine lumen, indicating that this could be a novel potential alternative therapy.
- Buffalo (Bubalus bubali) Late Embryo and Foetus Development: A
- Abstract: Many researches describe the embryonic developmental features in domestic animals; however, in farm animals, they are scarce. Most farm animal studies are related to assisted reproduction and embryos transfer techniques. But, morphological features and size measure to estimate the age gestation are rarely reported in literature. Thus, in this study, we described the developmental changes in the bubaline (Bubalus bubali) concepts from 21 to 60 days of gestation. Our results revealed that buffalo embryos similar morphological characteristics similar to other mammalian species. Also, similarities between bovine and bubaline persist; except on foetal stages when buffalos have a faster development than bovine. Therefore, buffalo's gestation period exhibits some varieties and accurate embryo age is more difficult. Yet, when we use a combination of the crown–rump, macroscopic analysis and alizarin red, it is possible to describe better the whole embryogenesis stages of the buffalo and which can contribute for future reproduction researches and applications in veterinary practice.
- Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine
Spermatozoa Subjected to Ferrous Ascorbate
- Abstract: Quercetin (QUE) is a natural flavonol‐type flavonoid with antibacterial, anti‐inflammatory and anti‐aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS‐mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 μmol/l) in the presence or absence of a pro‐oxidant, that is ferrous ascorbate (FeAA; 150 μmol/l FeSO4 and 750 μmol/l ascorbic acid) during a 6‐h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer‐aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p
- Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine
- Authors: CG Luchetti; RJ Bevacqua, MS Lorenzo, MF Tello, M Willis, CP Buemo, DM Lombardo, DF Salamone
Abstract: The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p
- Seasonal and Ageing‐Depending Changes of Aquaporins 1 and 9
Expression in the Genital Tract of Buffalo Bulls (Bubalus bubalis)
- Authors: S. Arrighi; G. Bosi, G. Accogli, S. Desantis
Abstract: The presence of Aquaporins 1 (AQP1) and 9 (AQP9), integral membrane water channels that facilitate rapid passive movement of water and solutes, was immunohistochemically detected in the excurrent ducts collected from sexually mature buffalo bulls of proven fertility during the mating (late autumn–winter) and non‐mating (late spring to the beginning of autumn) seasons. Furthermore, the research was performed also on the epididymal cauda of a senile buffalo bull with inactive testis. Aquaporins 1 and 9 were immunolocalized at distinct levels. In the efferent ducts, AQP1 immunoreactivity was strongly evidenced at the apical surface of the non‐ciliated cells and weakly along the basal membrane of the epithelial cells. The latter reactivity disappeared during the non‐mating season. No AQP1 immunoreactivity was detected in the epithelium of epididymis and vas deferens, whereas AQP1 was expressed in the smooth muscle layer of the vas deferens. Aquaporin 1 was present in the blood vessels and in small nerve bundles all along the genital tract. The supranuclear zone of the epididymal principal cells was AQP9 immunoreactive, limited to the corpus and cauda regions, and vas deferens. The samples collected in the two reproductive seasons showed a weaker AQP9 immunoreactivity during the non‐mating season. A typical AQP9 immunoreactivity was noticed in the old buffalo examined. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates, equine, dog and cat. In addition, seasonal differences were noticed which are possibly useful in regard to the comprehension of the morphophysiology of reproduction in the bubaline species, which are still a matter of debate.
- Sperm Morphology Assessment in Captive Neotropical Primates
- Abstract: The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO‐2010) standards and Sperm Deformity Index (SDI) criteria, objective computer‐assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p
- Fusion of Boar Sperm with Nanoliposomes Prepared from Synthetic
- Authors: VR Kasimanickam; MM Buhr
Abstract: Liposomes are artificial membrane vesicles that can be used to test and model the functions and interactions of various biological membranes, or as a carrier system to deliver biologically active substances into the cells, or to incorporate lipids into the plasma membrane of target cells to modify membrane structure–function relationships. Sperm plasma membrane undergoes lipid modification during maturation in epididymis and during capacitation in the female reproductive tract to facilitate fertilization. Natural variation in the amounts and composition of lipids in the sperm plasma membrane may also contribute to the species‐specific sperm sensitivities to handling and storage conditions. Boar sperm are notoriously susceptible to membrane damage and are resistant to compositional alteration by artificial liposomes. This study used flow cytometry to demonstrate stable incorporation of nanoliposomes prepared from a complex mixture of various phospholipids (phosphatidylcholine : phosphatidylethanolamine : sphingomyelin : phosphatidylserine : phosphatidylinositol) with high fusion efficiency. Over 90% of sperm rapidly took up fluorescently labelled liposomes and retained the lipids for at least 60 min, in a significant time‐ and concentration‐dependent manner. This unique fusion efficacy could be used to alter sperm plasma membrane composition and hence membrane‐based functional responses.
- Pregnancy Loss in Dairy Cattle: Relationship of Ultrasound, Blood
- Abstract: Objectives were to determine associations between percentage pregnancy loss (PPL) in dairy cattle and: (i) pregnancy diagnosis by ultrasonography; (ii) pregnancy diagnosis by serum pregnancy‐specific protein B (PSPB) concentrations, with or without serum progesterone concentrations; and (iii) production and environmental factors. This study included 149 822 pregnancy diagnoses conducted over 13 years in Holstein‐Friesian cows in Hungarian dairy herds. The following were determined: PPL in cows diagnosed pregnant by transrectal ultrasonography 29–42 days after artificial insemination (AI; n = 11 457); PPL in cows diagnosed pregnant by serum PSPB 29–35 days after AI (n = 138 365); and PPL and its association with serum progesterone concentrations, PSPB and production/environmental variables. The definition of PPL was percentage of cows initially diagnosed pregnant based on ultrasonography or PSPB, but not pregnant when examined by transrectal palpation 60 –70 days after AI. The PPL was lower (p
- First Production of Larvae Using Cryopreserved Sperm: Effects of
Preservation Temperature and Cryopreservation on European Eel Sperm
- Abstract: Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (‘cryolarvae’) were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.
- Serum MX2 Protein as Candidate Biomarker for Early Pregnancy Diagnosis in
- Authors: L Buragohain; R Kumar, T Nanda, SK Phulia, AK Mohanty, S Kumar, S Balhara, SPS Ghuman, I Singh, AK Balhara
Abstract: Interferon‐tau (IFN‐τ)‐induced molecular markers such as ubiquitin‐like modifier (ISG15), 2′,5′‐oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non‐inseminated (n = 8) and inseminated non‐pregnant (n = 10) control animals. In non‐inseminated and inseminated non‐pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post‐oestrus. However, in pregnant heifers, on 14th and 28th day post‐insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14–28 of pregnancy in buffalo holds potential for its use in early pregnancy detection.
- Can Microfiltered Seminal Plasma Preserve the Morphofunctional
Characteristics of Porcine Spermatozoa in the Absence of Antibiotics?
A Preliminary Study
- Authors: F Barone; D Ventrella, A Zannoni, M Forni, ML Bacci
Abstract: Artificial insemination is extensively performed in pig farms in Europe, the United States and Canada. Antibiotics are typically added to the inseminating dose to limit bacterial growth during liquid phase storage at 16°C, as bacterial contamination is unavoidable. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) take action to control and reduce antibiotic use in animals as more bacteria are becoming resistant to antimicrobials. To avoid the use of antibiotics, we prepared inseminating doses using microfiltered seminal plasma (SP). Microfiltration is a common technology used to reduce bacterial contamination but may retain seminal substances, influencing sperm quality during storage. Therefore, the aim of this study was to evaluate the morphofunctional parameters of spermatozoa during storage at 16°C in doses prepared with or without microfiltered SP, with or without the addition of antibiotics, in a Latin square design. Artificial insemination doses with microfiltered SP and without antibiotic addition preserved spermatozoa viability, mitochondrial membrane potential, acrosome integrity and objective motility, with absolute values equal or even better than those observed in conventional doses. In conclusion, although the results could be considered preliminary due to the small sample size, this study suggests that microfiltration of SP can be a simple method, feasible on farms, to replace antibiotic use in extended doses stored in the liquid phase at 16°C for up to 7 days.
- Effects of Inulin Supplementation in Low‐ or High‐Fat Diets on
Reproductive Performance of Sows and Antioxidant Defence Capacity in Sows
- Authors: YS Wang; P Zhou, H Liu, S Li, Y Zhao, K Deng, DD Cao, LQ Che, ZF Fang, SY Xu, Y Lin, B Feng, J Li, D Wu
Abstract: This experiment was conducted to investigate the effects of inulin supplementation in low‐ or high‐fat diets on both the reproductive performance of sow and the antioxidant defence capacity in sows and offspring. Sixty Landrace × Yorkshire sows were randomly allocated to four treatments with low‐fat diet (L), low‐fat diet containing 1.5% inulin (LI), high‐fat diet (H) and high‐fat diet containing 1.5% inulin (HI). Inulin‐rich diets lowered the within‐litter birth weight coefficient of variation (CV, p = 0.05) of piglets, increased the proportion of piglets weighing 1.0–1.5 kg at farrowing (p
- Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic
Sperm Injection in Caprine Oocytes
- Authors: SD Kharche; J Pathak, S Agarwal, B Kushwah, AKS Sikarwar
Abstract: The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.
- Impact of Buserelin Acetate or hCG Administration on the Day of First
Artificial Insemination on Subsequent Luteal Profile and Conception Rate
in Murrah Buffalo (Bubalus bubalis)
- Authors: AK Pandey; SPS Ghuman, GS Dhaliwal, SK Agarwal, JB Phogat
Abstract: This study was designed to investigate the impact of buserelin acetate (BA) or human chorionic gonadotropin (hCG) administration on the day of first artificial insemination (AI) on subsequent luteal profile (diameter of corpus luteum (CL) and plasma progesterone) and conception rate in Murrah buffalo. The present experiment was carried out at two locations in 117 buffalo that were oestrus‐synchronized using cloprostenol (500 μg) administered (i.m.) 11 days apart followed by AI during standing oestrus. Based on treatment (i.m.) at the time of AI, buffalo were randomly categorized (n = 39 in each group) into control (isotonic saline solution, 5 ml), dAI‐BA (buserelin acetate, 20 μg) and dAI‐hCG (hCG, 3000 IU) group. Out of these, 14 buffalo of each group were subjected to ovarian ultrasonography on the day of oestrus to monitor the preovulatory follicle and on days 5, 12, 16 and 21 post‐ovulation to monitor CL diameter. On the day of each sonography, jugular vein blood samples were collected for the estimation of progesterone concentrations. All the buffalo (n = 117) were confirmed for pregnancy on day 40 post‐ovulation. The conception rate was better (p
- Influence of Glutamine Supplementation on Motility and Fertilization
Success of Frozen–Thawed Persian Sturgeon (Acipenser persicus) Sperm
- Authors: MS Aramli; K Golshahi, RM Nazari, A Golpour, S Aramli
Abstract: Amino acids have an important biological role for the prevention of cell damage during cryopreservation. The aim of this study was to investigate the effects of glutamine on post‐thaw sperm motility and fertilization success in the Persian sturgeon (Acipenser persicus). Sperm collected from six fish was cryopreserved in extenders containing different glutamine concentrations (2.5, 5 and 10 mm). Sperm samples diluted at the ratio of 1 : 1 using the extenders were subjected to cryopreservation. After dilution, the sperm suspensions were sucked into 250‐μl straws; the straws were placed on the tray, frozen in nitrogen vapour and plunged into liquid nitrogen. Then, sperm were thawed in a water bath at 40°C for 5 s and used for analysis. Our results revealed that an increase in the concentration of glutamine caused a significant increase in the motility percentage, curvilinear velocity (VCL) and also fertilization success in the Persian sturgeon (p
- Genetic‐Quantitative Study of the First‐Service Pregnancy
Probability of Murrah Heifers
- Abstract: Because of the importance of reproduction in stock breeding systems, it is necessary to find selection criteria that increase reproductive efficiency. The aim of this study was to estimate genetic parameters for the probability of conception on first service (PROB) in Murrah heifers, and its association with other traits of economic interest [age at first calving (AFC), service period, calving interval and milk yield at 270 days], with the purpose of evaluating their use as selection criteria. Reproductive information and first lactation records of 1200 Murrah heifers were used to perform two‐trait analyses between PROB and the other characteristics. Bayesian inference was used to estimate the variance components, considering PROB as threshold and the other as linear factors. The results demonstrate that this trait has heritability of 0.15, indicating the possibility of a genetic gain by using it for selection. With respect to the genetic correlation estimates, the only high‐magnitude association was with AFC (−0.899), which is the current criterion indicating sexual precocity of females. In the light of the parameters estimated, the first‐service pregnancy rate is an alternative for indication of sexual precocity, although presenting a smaller genetic gain than the current standard AFC. Nevertheless, additional research should be conducted regarding this trait to assess the economic importance of its use in dairy buffalo production systems.
- The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm
- Abstract: The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H2O2 0 mm = H000; H2O2 50 mm = H050; H2O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p
- Effects of Interferon‐Tau and Steroids on Cytochrome P450 Activity
in Bovine Endometrial Epithelial Cells
- Authors: CL Gilfeather; CO Lemley
Abstract: The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon‐τ (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were treated with OT, IFN, a combination of OT+IFN or control (CON) media for 24 h. For the second experiment, cells were treated with E2, P4, a combination of E2 + P4 or CON media for 24 h. Treatments were performed in triplicate, and the experiment was repeated four times (n = 12 per treatment). Treatment with OT alone increased (p
- Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles
- Abstract: This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM+, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p
- An In Vitro Evaluation of Biochemical Processes Involved in
Lead‐Induced Changes on Ram Spermatozoa
- Abstract: Lead (Pb2+) is a toxic heavy metal which interferes with several physiological processes regulated by Ca2+, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb2+/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb2+ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb2+ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb2+ action. On the contrary, Pb2+‐incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb2+ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb2+ in spermatozoa; thus, the enhancement of Pb2+ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb2+ by spermatozoa. However, the increase of intracellular Pb2+ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.
- Effect of Heat Stress on Concentrations of Faecal Cortisol Metabolites in
- Abstract: The negative impact of heat stress on health and productivity of dairy cows is well known. Heat stress can be quantified with the temperature–humidity index (THI) and is defined as a THI ≥ 72. Additionally, animal welfare is affected in cows living under heat stress conditions. Finding a way to quantify heat stress in dairy cows has been of increasing interest over the past decades. Therefore, the objective of this study was to evaluate concentrations of faecal glucocorticoid metabolites [i.e. 11,17‐dioxoandrostanes (11,17‐DOA)] as an indirect stress parameter in dairy cows without heat stress (DOA 0), with heat stress on a single day (acute heat stress, DOA 1) or with more than a single day of heat stress (chronic heat stress, DOA 2). Cows were housed in five farms under moderate European climates. Two statistical approaches (approach 1 and approach 2) were assessed. Using approach 1, concentrations of faecal 11,17‐DOA were compared among DOA 0, DOA 1 and DOA 2 samples regardless of their origin (i.e. cow, unpaired comparison with a one‐way anova). Using approach 2, a cow was considered as its own control; that is 11,17‐DOA was treated as a cow‐specific factor and only paired samples were included in the analysis for this approach (paired comparison with t‐tests). In approach 1 (p = 0.006) and approach 2 (p = 0.038), 11,17‐DOA values of cows under acute heat stress were higher compared to those of cows without heat stress. Our results also indicate that acute heat stress has to be considered as a confounder in studies measuring faecal glucocorticoid metabolites in cows to evaluate other stressful situations.
- Apoptotic Cell Localization in Preantral and Antral Follicles in Relation
to Non‐cyclic and Cyclic Gilts
- Authors: D Phoophitphong; S Srisuwatanasagul, S Koonjaenak, P Tummaruk
Abstract: The objective of this study was to determine apoptotic cell localization in preantral and antral follicles of porcine ovaries. Additionally, the proportion of cells undergoing apoptosis was also compared between delayed puberty gilts and normal cyclic gilts. Ovarian tissues were obtained from 34 culled gilts with age and weight of 270.1 ± 3.9 days and 143.8 ± 2.4 kg, respectively. The gilts were classified according to their ovarian appearance as ‘non‐cyclic’ (n = 7) and ‘cyclic’ (n = 27) gilts. The terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay was used to determine apoptotic cell expression in different compartments of the ovarian tissue sections. All apparent preantral (n = 110) and antral (n = 262) follicles were evaluated using image analysis software. It was found that apoptotic cells were expressed in both granulosa (22.2%) and theca cell layers (21.3%) of the follicles in the porcine ovaries. The proportion of apoptotic cells in the granulosa layer in the follicles was positively correlated with that in the theca layer (r = 0.90, p 0.05) or theca cell layers (28.6% and 26.5%, p > 0.05). The proportion of apoptotic cells in non‐cyclic gilts was higher than cyclic gilts in both granulosa (31.7% and 22.6%, p
- Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal
Porcine Cumulus Oocyte Complexes
- Abstract: Very small follicles (
- Pre‐Selection Test to Identify High Responder Donor Goats
- Abstract: The aim of the study was to evaluate the feasibility of pre‐selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long‐term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple‐dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG‐treated in comparision with FSH‐treated does (p
- Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions
- Abstract: Predicting male fertility on non‐invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer‐assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3–5, 6–10, 11–14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real‐time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.
- Morphology and Aquaporin Immunohistochemistry of the Uterine Tube of
- Authors: S Arrighi; G Bosi, S Frattini, B Croizet, D Groppetti, A Pecile
Abstract: The expression of six different aquaporins (AQP1, 2, 3, 4, 5 and 9), integral membrane water channels that facilitate bi‐directional passive movement of water, was investigated by immunohistochemistry in the uterine tube of pre‐pubertal and adult Saanen goats (Capra hircus), comparing the different phases of the oestrous cycle. Regional morphology and secretory processes were markedly different during the goat oestrous cycle. The tested AQP molecules showed different expression patterns in comparison with already studied species. AQP1‐immunoreactivity was evidenced at the endothelium of blood vessels and in nerve fibres, regardless of the tubal tract and cycle period. AQP4‐immunoreactivity was shown on the lateral plasmalemma in the basal third of the epithelial cells at infundibulum and ampulla level in the cycling goats, more evidently during follicular than during luteal phase. No AQP4‐immunoreactivity was noticed at the level of the isthmus region, regardless of the cycle phase. AQP5‐immunoreactivity, localized at the apical surface of epithelial cells, increased from pre‐puberty to adulthood. Thereafter, AQP5‐immunoreactivity was prominent during the follicular phase, when it strongly decorated the apical plasmalemma of all epithelial cells at ampullary level. During luteal phase, immunoreactivity was discontinuous, being weak to strong at the apex of the secretory cells protruding into the lumen. In the isthmus region, the strongest AQP5‐immunoreactivity was seen during follicular phase, with a clear localization in the apical plasmalemma of all the epithelial cells and also on the lateral plasmalemma. AQP2, 3 and 9 were undetectable all along the goat uterine tube. Likely, a collaboration of different AQP molecules sustains the fluid production in the goat uterine tube. AQP1‐mediated transudation from the blood capillaries, together with permeation of the epithelium by AQP4 in the basal rim of the epithelial cells and final intervening of apical AQP5, could be involved in fluid production as well as in secretory processes.
- Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6
- Abstract: The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.
- Serum Levels of Cardiac Markers NT‐proANP and NT‐proBNP in
Brachycephalic bitches at Different Gestational Stages
- Authors: MAR Feliciano; RR Uscategui, GS Maciel, VT Almeida, MF Silveira, GAC Oliveira, WRR Vicente
Abstract: The aim of this study was to determine serum levels of natriuretic peptide precursors (NT‐proANP and NT‐proBNP) during pregnancy in brachycephalic bitches. Fifteen healthy multiparous bitches were selected for this prospective study. Serum levels of NT‐proANP and NT‐proBNP were measured during anoestrous and at 14, 35, 42, 49 and 56 days (2nd, 5th, 6th, 7th and 8th weeks) of pregnancy. Fourteen animals had normal gestations, and one bitch developed single foetus syndrome. The natriuretic peptide levels of this animal were not included in this study; however, it is important to report that its NT‐proANP levels were four times greater than those of normal patients. There was no significant difference (p = 0.072) in NT‐proBNP levels between anoestrous (0.20 ± 0.10 ng/ml) and the different pregnancy weeks (0.27 ± 0.12 ng/ml). There was a positive correlation (p
- Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass
in In Vitro Obtained Cats' Blastocysts
- Abstract: The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre‐implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.
- Issue Information
- Pages: 337 - 338
- Hepatitis B Surface Antigen S Gene is an Effective Carrier Molecule for
Developing GnRH DNA Immunocastration Vaccine in Mice
- Authors: YG Han; WJ Ye, GQ Liu, XP Jiang, N Ijaz, JY Zhao, B Tesema
Pages: 445 - 450
Abstract: Relatively molecular mass of GnRH antigens is small and hence needs to couple to a large carrier molecule to enhance its immunogenicity. This study investigated whether hepatitis B surface antigen S (HBsAg‐S) gene can be used as an effective carrier molecule for developing GnRH DNA immunocastration vaccine. Two copies of human GnRH gene were fused with HBsAg‐S gene for constructing a recombinant plasmid pVAX‐HBsAg‐S‐2GnRH that coded for 27 kDa target fusion protein. Ten male mice were divided into two equal groups, treatment and control. The vaccine (50 μg/mice) prepared in saline solution was injected into male mice at weeks 0, 1, 2, 4 and 7 of the experiment. Vaccine's efficacy was evaluated in terms of GnRH‐specific IgG antibody response, plasma testosterone levels, testicular weight and extent of the testicular tissue damage. The specific anti‐GnRH antibody titre in vaccinated animals was significantly higher than in controls in only 4th week of immunization (p