- Are Pharmacological Interventions Between Conception and Birth Effective
in Improving Reproductive Outcomes in North American Swine'
- Authors: JM Wessels; K Khalaj, RT Kridli, AK Edwards, M Bidarimath, C Tayade
Abstract: The objective of this review is to evaluate the effectiveness of using pharmacological compounds on reproductive outcomes, particularly litter size, in North American swine. While the opportunity to improve reproduction in North American pigs exists, numerous hurdles need to be overcome in order to achieve measureable results. In the swine industry, the majority of piglet losses are incurred during pregnancy and around farrowing. Over the last 20 years, a reduction in losses has been achieved through genetic selection and nutritional management; however, these topics are the focus of other reviews. This review will evaluate attempts to improve litter size by reducing losses at various stages of the reproductive process, from the time of conception to the time of farrowing, using pharmacological compounds. Generally, these compounds are used to either alter physiological processes related to fertilization, embryonic attachment or uterine capacity, etc., or to facilitate management aspects of the breeding females such as inducing parturition. Although some of the pharmacological agents reviewed here show some positive effects on improving reproductive parameters, the inconsistent results and associated risks usually outweigh the benefits gained. Thus, at the present time, the use of pharmacological agents to enhance reproduction in North American swine may only be recommended for herds with low fertility and presents an avenue of research that could be further explored.
- Systemic Immunosuppression by Methylprednisolone and Pregnancy Rates in
Goats Undergoing the Transfer of Cloned Embryos
- Authors: C Feltrin; CA Cooper, N Mohamad-Fauzi, VHV Rodrigues, LH Aguiar, S Gaudencio-Neto, LT Martins, CEM Calderón, AS Morais, IS Carneiro, TM Almeida, ING Silva, JL Rodrigues, EA Maga, JD Murray, AB Libório, LR Bertolini, M Bertolini
Abstract: The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.
- Comparison of Cryoprotective Effects of Lycopene and Cysteamine in
Different Cryoprotectants on Bull Semen and Fertility Results
- Authors: PB Tuncer; S Büyükleblebici, A Eken, U Taşdemir, E Durmaz, O Büyükleblebici, E Çoşkun
Abstract: The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty-four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5 mm or lycopene 500 μg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non-return rates of inseminated cows. However, this result was not considered to be statistically important.
- Forced Collapse of the Blastocoel Cavity Improves Developmental Potential
in Cryopreserved Bovine Blastocysts by Slow-Rate Freezing and
- Authors: S-H Min; J-W Kim, Y-H Lee, S-Y Park, P-S Jeong, J-Y Yeon, H Park, K-T Chang, D-B Koo
Abstract: This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow-rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow-rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non-FBC groups were found in both slow-rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real-time RT-PCR analysis data showed that expression of the anti-apoptotic Bcl-XL gene was significantly increased by FBC groups, whereas expression of the pro-apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow-rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre-treatment technique for both slow-rate freezing and vitrification of bovine blastocysts.
- Husbandry Factors and the Resumption of Luteal Activity in Open and
Zero-Grazed Dairy Cows in Urban and Peri-Urban Kampala, Uganda
- Authors: BM Kanyima; R Båge, DO Owiny, T Ntallaris, J Lindahl, U Magnusson, MG Nassuna-Musoke
Abstract: The study investigated the influence of selected husbandry factors on interval to resumption of post-partum cyclicity among dairy cows in urban and peri-urban Kampala. A prospective study of 85 day post-partum period of 59 dairy cows in open (n = 38) and zero grazing (n = 21) systems was conducted on 24 farms. Cows of parity 1–6 were recruited starting 15–30 days post-partum. Progesterone (P4) content in milk taken at 10–12 day intervals was analysed using ELISA. The cow P4 profiles were classified into ‘normal’ ( 56 days), ‘ceased’ or ‘prolonged’ (if started
- The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty
on Male Wistar Rats
- Authors: S Caceres; G Silvan, L Martinez-Fernandez, MJ Illera, P Millan, B Monsalve, L Peña, JC Illera
Abstract: Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p
- Caspase Activation, Hydrogen Peroxide Production and Akt Dephosphorylation
Occur During Stallion Sperm Senescence
- Authors: JM Gallardo Bolaños; C Balao da Silva, P Martín Muñoz, M Plaza Dávila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola, FJ Peña
Abstract: To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.
- Impact of Probing the Reproductive Tract During Early Pregnancy on
Fertility of Beef Cows
- Authors: G Pugliesi; SC Scolari, FS Mesquita, M Maturana Filho, ER Araújo, D Cardoso, JN Sales, I Martin, M Sá Filho, CM Bertan, M Binelli
Abstract: This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.
- An Immunohistochemical Observations on the Oviduct of the Goat
- Authors: E Marettová; M Maretta
Abstract: The oviduct has an important role in regulating transport of gametes and fertilization. The main role in these functions has a smooth muscle cells and ciliated epithelium lining the oviduct. All functions are under the influence of hormonal and nervous system. The objective of this study was immunohistochemically to examine the following structures: lining epithelium, smooth muscle cells, elastic fibres and nerve fibres. For this purpose, the following antibodies were used: cytokeratin 18, S-100 protein, acetylated α-tubulin, smooth muscle actin, desmin and elastin. Ciliary and secretory cells of the lining epithelium were positive for cytokeratin 18 and S-100 protein. Cilia and the basal body-associated structures of ciliary cells were positive to acetylated α-tubulin. Smooth muscle cells (SMC) in mucosa and of the muscular layer were positive for α-smooth muscle actin (SMA) and desmin. High density of nerve fibres positively reacted to acetylated α-tubulin and S100 protein was present in the mucosa, muscular layer and serosa. Elastic fibres positive for elastin form a dense network at the base of the mucosal folds and in the muscle layer. A dense network of these fibres is accompanying the blood vessels. It is supposed that together with smooth muscle cells they are involved in the transport of ovum and in blood flow regulation.
- Apoptosis-Like Events and In Vitro Fertilization Capacity of Sex-Sorted
- Authors: X-M Zhao; J-J Ren, S-J Zhao, L-S Cui, H-S Hao, H-Y Wang, W-H Du, T Qin, Y Liu, D Wang, H-B Zhu
Abstract: This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC-1) and TUNEL) for flow cytometric analysis of apoptosis in sex-sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (∆ψm) showed that the sex-sorted sperm collected from bull A significantly differed from those of the other three bulls (p
- Measurement of C-reactive protein and Prostaglandin F2α Metabolite
Concentrations in Differentiation of Canine Pyometra and Cystic
- Authors: SO Enginler; A Ateş, B Diren Sığırcı, BH Sontaş, K Sönmez, E Karaçam, H Ekici, G Evkuran Dal, A Gürel
Abstract: Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C-reactive protein (CRP) and prostaglandin F2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p
- Kinematics and Subpopulations’ Structure Definition of Blue Fox
(Alopex lagopus) Sperm Motility using the ISAS® V1 CASA System
- Authors: C Soler; A García, J Contell, J Segervall, M Sancho
Abstract: Over recent years, technological advances have brought innovation in assisted reproduction to the agriculture. Fox species are of great economical interest in some countries, but their semen characteristics have not been studied enough. To advance the knowledge of function of fox spermatozoa, five samples were obtained by masturbation, in the breeding season. Kinetic analysis was performed using ISAS® v1 system. Usual kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were considered. To establish the standardization for the analysis of samples, the minimum number of cells to analyse and the minimum number of fields to capture were defined. In the second step, the presence of subpopulations in blue fox semen was analysed. The minimum number of cells to test was 30, because kinematic parameters remained constant along the groups of analysis. Also, the effectiveness of ISAS® D4C20 counting chamber was studied, showing that the first five squares presented equivalent results, while in the squares six and seven, the kinematic parameters showed a reduction in all of them, but not in the concentration or motility percentage. Kinematic variables were grouped into two principal components (PC). A linear movement characterized PC1, while PC2 showed an oscillatory movement. Three subpopulations were found, varying in structure among different animals.
- Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes
- Authors: E Dovolou; IE Messinis, E Periquesta, K Dafopoulos, A Gutierrez-Adan, GS Amiridis
Abstract: Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.
- Effect of Exposure to Heatwave During Blastocyst Formation on Reproductive
Performance of Female Rabbits
- Authors: F Marco-Jiménez; C Naturil-Alfonso, DS Peñaranda, E Jiménez-Trigos, FJ García-Diego, JS Vicente
Abstract: We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14–22°C, n = 29) and does heat stressed in a climatic chamber (maintained between 32–37°C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-ɣ, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8–16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation.
- Evaluation of Oestrous Detection in Dairy Cattle Comparing an Automated
Activity Monitoring System to Visual Observation
- Authors: I Michaelis; O Burfeind, W Heuwieser
Abstract: The objective of the study was to evaluate an automated activity monitoring (AAM) system for oestrous detection in dairy cows. Specifically, we set out to determine the oestrous detection efficiency and accuracy of the AAM system and to compare reproductive performance of cows detected either by AAM, by visual observation (VO) or by at least 1 of the 2 methods (EOM). A total of 1004 potential cow-periods from 348 cows were analysed. Oestrous detection rates (EDR) were calculated for 21 day (VWP+21), 42 day (VWP+42), and 63 day (VWP+63) after voluntary waiting period (VWP). Oestrous detection rate did not differ between AAM (42.1%) and VO (37.3%) during VWP+21 (p > 0.05) but was significantly higher in EOM (56.3%, p
- Pregnancy-Associated Glycoprotein (PAG) Profile of Holstein–Friesian
Cows as Compared to Dual-Purpose and Beef Cows
- Authors: M Shahin; M Friedrich, M Gauly, W Holtz
Abstract: Pregnancy-associated glycoproteins (PAGs) are produced by mono- and binucleate trophoblast cells in the ruminant placenta. PAG appears in maternal blood and, from approximately 4 weeks after fertilization onward, may serve as a reliable means of diagnosing pregnancy. A range of factors are said to affect plasma PAG concentrations, such as number and sex of foetus, mass of calf and placenta, level of milk production and genetic constitution. In this study, PAG pregnancy profiles of a dual-purpose (Simmental) and two beef breeds (Uckermark and Aubrac) are compared with the profile of the specialized dairy breed Holstein–Friesian. Holstein–Friesian cows were sampled weekly; the levels of the other breeds were presented at 3-week intervals. The overall significant breed difference (p = 0.013) was founded on deviations during the initial 3 weeks of pregnancy and from 23 weeks onward. During the period critical for the detection of pregnancy, between four and 22 weeks, agreement between PAG levels of various breeds was close (p > 0.05). No significant effect of body mass of cow or calf (relative to mass of dam) was detected. These findings imply that the PAG pregnancy test may be executed uniformly irrespective of breed or type of cow, affirming the suitability of the test as a valuable asset for the cattle industry.
- Coexistence of Tuberculosis and Mammary Carcinoma in a Goat
- Authors: H Quintas; N Alegria, A Mendonça, A Botelho, A Alves, I Pires
Abstract: Synchronic occurrence of tuberculosis mastitis and mammary cancer is rare in humans and, to the best of our knowledge, not reported in domestic animals. Here, we present a case of a female adult goat of Serrana breed with simultaneous occurrence of a granulomatous mastitis, due to Mycobacterium caprae, and a mammary carcinoma. Both pathological conditions are rare in goats and should be included in differential diagnosis of mammary lesions.
- ATP Content, Oxidative Stress and Motility of Beluga (Huso huso) Semen:
Effect of Short-Term Storage
- Authors: MS Aramli
Abstract: An effective technique for short-term storage of semen is essential when processing multiple sperm samples and when semen must be transported from collection sites to hatcheries for the fertilization of ova or to laboratories for cryopreservation. In this experiment, beluga (Huso huso) sperm were used to evaluate the effects of short-term storage on several quality parameters (i.e. motility, adenosine triphosphate (ATP) content and oxidative stress indices). Sperm cells exhibited> 50% motility during 3 days of storage with an average total duration of sperm motility varying from 13.33 ± 5.77 to 278.33 ± 25.65 s, and no motile spermatozoa were recorded after 9 days of storage. The levels of oxidative stress indices (thiobarbituric acid reactive substances and carbonyl derivatives of proteins) and antioxidant activity (superoxide dismutase) increased significantly after 3 days of storage. The ATP content also decreased significantly after 2 days of storage. The results of this study can be used to develop effective reproduction management and cryopreservation protocols for this endangered fish.
- Genetic Parameters and Breeding Values for Semen Characteristics in
- Authors: D Labitzke; H Sieme, G Martinsson, O Distl
Abstract: The objectives of this study were to show whether semen traits of 30 Hanoverian stallions regularly used in AI may be useful for breeding purposes. Semen characteristics were studied using 15 149 ejaculates from 30 Hanoverian stallions of the State Stud Celle of Lower Saxony. Semen samples were collected between 2005 and 2009. Traits analysed were gel-free volume, sperm concentration, total and motile sperm number and progressive motility. A linear multivariate animal model was employed to estimate heritabilities and permanent environmental variances for stallions. The same model was used to predict breeding values for all traits simultaneously. Heritabilities were high for gel-free volume (h2 = 0.43) and moderate for total number of sperm (h2 = 0.29) and progressive motility (h2 = 0.20). Gel-free volume, sperm concentration and total number of sperm were genetically negatively correlated with progressive motility. The effect of the permanent environment for stallions accounted for 9–55% of the trait variance. The total variance among stallions explained 37–69% of the trait variance. The average reliabilities of the breeding values were 0.43–0.76 for the 30 Hanoverian stallions. In conclusion, the study could demonstrate large effects of stallions, routinely employed in a breeding programme, on semen characteristics analysed here. We could demonstrate that estimated breeding values (EBV) with sufficient high reliabilities can be predicted using data from these stallions and these EBV are useful in horse breeding programmes to achieve genetic improvement in semen quality.
- The Influence of Morphology, Follicle Size and Bcl-2 and Bax Transcripts
on the Developmental Competence of Bovine Oocytes
- Authors: THC De Bem; PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles, CLV Leal
Abstract: This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1–3 mm (Bax; 1.0 and Bcl-2; 1.0), 3–6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6–9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles>3 mm in diameter are better suited to in vitro embryo development.
- A Case of Leucocyte Chimerism (78,XX/78,XY) in a Dog with a Disorder of
- Authors: I Szczerbal; J Nowacka-Woszuk, W Nizanski, S Salamon, M Ochota, S Dzimira, W Atamaniuk, M Switonski
Abstract: A 1-year-old Shih Tzu dog was presented for examination because of abnormal external genitalia. A residual penis with a prepuce was located in a position typical of a male. The dog had no palpable testicles or scrotum. The ultrasound examination revealed the presence of the prostate, but the gonads remained undetectable. Cytogenetic analysis performed on chromosome preparations obtained from lymphocyte culture showed two cell lines – 78,XX and 78,XY. Molecular analysis of 14 polymorphic microsatellite markers allowed us to distinguish leucocyte chimerism from whole body chimerism. The presence of 3 or 4 alleles was confirmed in DNA isolated from blood, while in DNA isolated from hair follicles only 1 or 2 alleles were detected. The case was classified as leucocyte 78,XX/78,XY chimerism. Our study showed that XX/XY leucocyte chimerism might be associated with disorder of sexual development in dogs. Furthermore, it is emphasized that the use of cytogenetic study, in combination with analysis of polymorphic markers in DNA isolated from different somatic cells, facilitates distinguishing between leucocyte and whole body chimerism.
- Dietary Fat Supplementation and the Consequences for Oocyte and Embryo
Quality: Hype or Significant Benefit for Dairy Cow Reproduction'
- Authors: JLMR Leroy; RG Sturmey, V Van Hoeck, J De Bie, PJ McKeegan, PEJ Bols
First page: 353
Abstract: In many countries, fat supplementation in the diet has become common in the dairy industry. There are several ideas as to how dietary fat could influence reproductive performance. Saturated fatty acids, such as palm oil, can increase milk yield but may aggravate negative energy balance and thus may impair fertility when fed during the first week post-partum. However, priming the lipid oxidation in the liver by feeding saturated fats during the dry period has recently been shown to be a potentially promising strategy to mitigate fat mobilization and liver accumulation post-partum. Furthermore, polyunsaturated fats (omega-3 fatty acids and conjugated linoleic acids) are fed to reduce the ‘de novo’ fat synthesis in the udder and thus the milk fat content, which may be of modest benefit for overall energy balance. Furthermore, omega-6 and omega-3 polyunsaturated fatty acids are reported to alter follicular growth, steroid synthesis and prostaglandin metabolism in the ovary and endometrium, respectively. Omega-6 fatty acids are believed to have pro-inflammatory and thus PGF2α-stimulating properties rendering them extra value as ‘nutraceutical’ early post-partum, while omega-3 fatty acids can weaken this inflammatory potency, leading to a higher chance of survival of the embryo when supplemented during the periconceptual period. Unfortunately, research results rarely provide a consensus in this perspective. The consequences of these fat-feeding strategies on oocyte and embryo quality remain an intriguing issue for debate. Fat feeding may alter the microenvironment of the growing and maturing oocyte of the early and older embryo and thus may affect reproductive outcome. We recently reported that dietary-induced hyperlipidaemic conditions can be harmful for embryo development and metabolism. However, to date, research results remain somewhat conflicting most probably due to differences in fat sources used, in diet and duration of supplementation and in experimental set-up in general.
- The Use of Liveweight Change as an Indicator of Oestrus in a Seasonally
Calving, Pasture-Fed Dairy Herd
- Authors: JI Alawneh; MA Stevenson, NB Williamson, N Lopez-Villalobos
First page: 362
Abstract: This was an observational study of 828 lactations in 542 mixed-age dairy cows that calved seasonally in a single, pasture-fed herd in New Zealand in 2008 and 2009. The study objectives were to: (i) document daily liveweight change (∆LW) before and after observed oestrus for cows subsequently diagnosed pregnant or non-pregnant and (ii) quantify the sensitivity and specificity of ∆LW as a test for oestrus. The sensitivity and specificity of ∆LW when combined with other commonly used oestrous detection methods was also evaluated. In cows that conceived as a result of service at detected oestrus, liveweight loss began 1 day before the day of detection and was greatest on the day of detection (−9.6 kg, 95% CI −11.3 kg to −7.8 kg; p
- Effect of Bovine Follicular Fluid on Reactive Oxygen Species and
Glutathione in Oocytes, Apoptosis and Apoptosis-related Gene Expression of
In vitro-Produced Blastocysts
- Authors: S-H Park; H-S Cho, I-J Yu
First page: 370
Abstract: The reactive oxygen species (ROS) generated during the in vitro maturation of oocytes affect oocyte maturation and subsequent embryonic development. Bovine follicular fluid (bFF) has an effective antioxidant capacity. This study was conducted to investigate the effects of supplementing oocyte maturation media with bFF from different size classes (3–8 and 9–13 mm) on the glutathione (GSH) and ROS levels of oocytes. Embryonic development and apoptosis, as well as the relative abundance of INFτ, BAX, BCL2 and HSP70 transcripts in blastocysts, were also monitored. Oocytes collected from ovaries were matured in TCM-199 with FBS (control) and 10% 3–8 mm (M), 9–13 mm (L) or a mixture of 3–8 mm and 9–13 mm (M + L) bFF. Glutathione and ROS levels in oocytes after 24 h were assessed by Cell Tracker Blue CMF2HC and DCHFDA staining, respectively. Apoptosis in day-8 blastocysts was assessed by TUNEL staining. The relative abundance of BAX, BCL2, HSP70 and INFτ transcripts was assessed using quantitative real-time polymerase chain reaction (PCR). The GSH level was significantly higher in the L group compared to the other groups (p
- Expression of Adiponectin and its Receptors in the Porcine Hypothalamus
During the Oestrous Cycle
- Authors: T Kaminski; N Smolinska, A Maleszka, M Kiezun, K Dobrzyn, J Czerwinska, K Szeszko, A Nitkiewicz
First page: 378
Abstract: Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p
- Seasonal Variations in Seminal Plasma Proteins of Buffalo
- Authors: L Sharma; V Pandey, R Nigam, P Singh, A Saxena, DK Swain
First page: 387
Abstract: The study was designed to evaluate the influence of season on semen characteristics and seminal plasma protein profile of buffalo bull semen. Thirty-six ejaculates were collected in three seasons (winter, summer and rainy) from six adult Bhadawari bulls, and semen characteristics were evaluated immediately after collection. The seminal plasma was harvested by centrifugation and protein profiling, and percentage protein fractions were analysed by SDS-PAGE. The significant effect of season was observed on ejaculate volume, sperm concentration, progressive motility, percentage live spermatozoa, hypo-osmotic swelling test (HOST) and acrosomal integrity. The electrophoretogram of seminal plasma proteins revealed 20 protein bands in winter, 23 bands in rainy and 25 bands in summer seasons, illustrating the significant effect of seasons on seminal plasma proteins. Among these protein bands, 18 bands were observed common in semen samples of all three seasons while protein bands of 46, 55, 58, 144 and 160 kDa were found in rainy and summer seasons. The protein bands of 48 and 60 kDa were observed only in winter season, whereas 184 and 200 kDa were reported in summer season only. The protein fractions (protein%) of common protein bands observed in three seasons revealed a significant effect of season on protein bands of 24.5, 66, 70, 72, 84 and 86 kDa. From the study, it was pertinent that bull seminal plasma contains specific proteins in particular season, which may be associated with some of the semen characteristics, and these proteins could be used as markers of the semen quality of buffalo bulls.
- Acute and Chronic Effects of a Contraceptive Compound RTI-4587-073(l) on
Testicular Histology and Endocrine Function in Miniature Horse Stallions
- Authors: MA Pozor; G Zambrano, J Roser, R Hess, S Runyon, E Runcan, BF Thomas, D Dymock, ML Macpherson, MH Troedsson, A Kelleman
First page: 392
Abstract: The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI-4587-073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI-4587-073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p
- IFN-τ Acts in a Dose-Dependent Manner on Prostaglandin Production by
Buffalo Endometrial Stromal Cells Cultured in vitro
- Authors: SG Chethan; SK Singh, J Nongsiej, HB Rakesh, RP Singh, N Kumar, SK Agarwal
First page: 403
Abstract: Interferon-τ (IFN-τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2). PGF2α is responsible for the luteolysis; however, PGE2 favours establishment of pregnancy by its luteoprotective action. In this study, the dose-response effect of recombinant bovine IFN-τ (rbIFN-τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN-τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN-τ treatment enhanced the secretion of both PGE2 and PGF2α, and also its ratio in a strict dose-dependent manner with a significant increase (p
- Parameters of the Reproductive Tract, Spermatogenesis, Daily Sperm
Production and Major Seminal Plasma Proteins of Tropically Adapted Morada
- Authors: FML Sousa; CH Lobo, ESB Menezes, JPA Rego, RV Oliveira, AC Lima-Souza, M Fioramonte, FC Gozzo, RCFF Pompeu, MJD Cândido, JT Oliveira, AA Moura
First page: 409
Abstract: This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42 weeks of age, 15 rams were subjected to semen collection and, 1 week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5 ± 0.5 cm and 109.5 ± 6.0 g, respectively. Seminiferous tubule (ST) diameter was 188.3 ± 4.0 μm and each testis contained 1.9 ± 0.1 Sertoli cells (×109). Each Sertoli cell supported 0.1 ± 0.01 A spermatogonia, 3.0 ± 0.2 pachytene spermatocytes and 7.7 ± 0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6 × 106 cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9 ± 9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22 kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology.
- Luteoprotective Role of Equine Chorionic Gonadotropin (eCG) During
Pregnancy in the Mare
- Authors: G Flores-Flores; E Velázquez-Cantón, M Boeta, L Zarco
First page: 420
Abstract: The effects of repeated cloprostenol administration were compared in mares impregnated by horses and mares impregnated by donkeys in order to assess the role of eCG on the development of pregnancy-associated resistance to the luteolytic and abortifacient effects of PGF2α. Eleven mares impregnated by donkey (mule pregnancy) and 9 mares impregnated by horse (horse pregnancy) were used. Six mares with mule pregnancy and four with horse pregnancy were injected with cloprostenol (0.25 mg) when they were between day 65 and day 75 of pregnancy, and the treatment was repeated 48, 72 and 96 h latter. The rest of the mares remained as controls. Concentrations of eCG were 10 times higher (p
- Addition of Cholesterol-Loaded Cyclodextrins to the Thawing Extender:
Effects on Boar Sperm Quality
- Authors: C Tomás; J Gómez-Fernández, E Gómez-Izquierdo, E Mocé, E Mercado
First page: 427
Abstract: The aim of the present study was to evaluate the effect that the addition of cholesterol-loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen-thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg-yolk-based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 106 sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p
- Kisspeptin, c-Fos and CRFR Type 2 Co-expression in the Hypothalamus after
- Authors: C Fergani; JE Routly, DN Jones, LC Pickavance, RF Smith, H Dobson
First page: 433
Abstract: Normal reproductive function is dependent upon availability of glucose and insulin-induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c-Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p
- Immunolocalization of the Cholesterol Transporters ABCA1 and ABCG1 in
Canine Reproductive Tract Tissues and Spermatozoa
- Authors: N Palme; AC Becher, M Merkl, M Glösmann, C Aurich, S Schäfer-Somi
First page: 441
Abstract: The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP-binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.
- Plasma Antimullerian Hormone as a Predictor of Ovarian Antral Follicular
Population in Bos indicus (Nelore) and Bos taurus (Holstein) Heifers
- Authors: EOS Batista; GG Macedo, RV Sala, MDDV Ortolan, MF Sá Filho, TA Del Valle, EF Jesus, RNVR Lopes, FP Rennó, PS Baruselli
First page: 448
Abstract: In Bos taurus cattle, antimullerian hormone (AMH) has been demonstrated to have a high degree of correlation with ovarian antral follicle count and the number of healthy follicles and oocytes. To document the correlation between the plasma concentration of AMH and follicular number in Bos indicus and Bos taurus heifers, Nelore (Bos indicus, n = 16) and Holstein heifers (Bos taurus, n = 16) had their ovarian follicular waves synchronized. After synchronization, ovarian antral follicular population (AFP) was evaluated three times at 60-day (d) intervals (T-120 d, 120 days before plasma AMH determination; T-60 d, 60 days before; and T0, at the time of plasma AMH determination). The plasma AMH concentration was positively correlated with the number of ovarian follicles on the day of the follicular wave emergence in Bos indicus (Nelore) and Bos taurus (Holstein) heifers at each evaluation time (p
- Treatment with eCG Decreases the Vascular Density and Increases the
Glandular Density of the Bovine Uterus
- Authors: J Mona e Pinto; V Pavanelo, L Alves de Fátima, LM Medeiros de Carvalho Sousa, G Pacheco Mendes, R Machado Ferreira, H Ayres, P Sampaio Baruselli, F Palma Rennó, P Carvallo Papa
First page: 453
Abstract: The uterus plays an essential role in mammalian reproduction and is a target of several hormonal protocols used to improve fertility in cattle. Many studies highlighted the importance of eCG treatment following fixed-time artificial insemination in improving follicular growth, ovulation and pregnancy rates in cattle. Moreover, eCG has been implicated in angiogenesis, leading to important changes in uterine blood flow and vascularisation. However, there is still a lack of information regarding the specific alterations induced by eCG upon glandular and vascular characteristics of bovine uterus. To investigate the influence of eCG on: uterine thickness and area; uterine artery diameter and area; uterine vascular and gland density; and the expression of the VEGFA-system, the uteri of crossbred beef cows were collected. All cows were submitted to follicular wave emergence synchronization. On day four of protocol, cows submitted to superovulation (n = 6) received 2000 IU eCG, on day eight, after expected follicular deviation, cows submitted to stimulatory treatment (n = 5) received 400 IU eCG. Control cows (n = 5) did not receive eCG. On day five po cows were subjected to ultrassonographic evaluation and slaughtered for uterine tissue sampling on day six po. Uterine vessels and glands were quantified by the counting point stereological method. The VEGFA-system was localized in different cellular types, showing no qualitative or quantitative differences in the site of expression or the intensity of the positive signal among the groups. Vascular density was decreased in the endometrium of stimulated and myometrium of superovulated cows compared with the control ones, which showed higher vascular density in the myometrium and endometrium of the ipsilateral uterine horn. The uterine gland density was higher in superovulated compared with stimulated and control cows. Thus, we can infer that stimulatory or superovulatory treatments with eCG influence the vascular density in the endometrium and myometrium in cattle.
- Gross Pathology and Endocrinology of Ovarian Cysts in Bitches
- Authors: Y Knauf; H Bostedt, K Failing, S Knauf, A Wehrend
First page: 463
Abstract: A total of 73 bitches with ovarian cysts were ovariohysterectomized. Cysts were characterized by gross pathology and endocrine parameters. Therefore, oestradiol-17ß and progesterone concentrations were assessed in cyst-fluid and corresponding blood plasma in each bitch. Our data demonstrated that multiple cysts were often present in a single individual (82%) and that cysts were commonly found on both ovaries (77%). The number of cysts per individual varied from 1 to 35. Most cysts were small in size (range 0.2–4.0 cm in diameter). No cyst was found to produce solely oestradiol-17ß or progesterone. Plasma levels of oestradiol-17ß and progesterone for a given individual were positively correlated with levels of these same hormones in their cyst-fluid (r = 0.334 and p = 0.001 for oestradiol-17ß; r = 0.419 and p
- Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence
Related to Their in vitro Maturation
- Authors: M Jeseta; D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka, M Machatkova
First page: 469
Abstract: This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.
- Effect of Leptin on In Vivo Goat Embryo Production
- Authors: AM Batista; WA Gomes, CCD Carvalho, PLJ Monteiro, FLM Silva, FC Almeida, PC Soares, GF Carneiro, MMP Guerra
First page: 476
Abstract: The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle-stimulating hormone (FSH) i.m. in six descending doses at 12-h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate-buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick-end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E2) and progesterone (P4) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p
- Integrated Evaluation of Scrotal Temperature and Testosteronemia after
GnRH Administration in Young Bulls with Low Semen Production
- Authors: J Vencato; L Cestaro, I Vazzana, G Carrer, E Carlo, S Dara, C Stelletta
First page: 481
Abstract: The aim of this study was to determine the suitability of thermographic monitoring of scrotal surface temperature (SST) as a method to monitor testicular function. Yearling bulls (n = 23) with low semen production were selected. Scrotal surface temperature and serum testosterone (T) concentrations were evaluated before and after administration of 10.5 μg buserelin acetate IV. Thermographic images of scrotum were recorded at 0, 15, 30, 45 and 60 min post-GnRH, while blood sampling was only performed at 60 min post-GnRH. Bulls were divided in two groups: LowTemp bulls (n = 10) had a decreased SST at 60 min; HighTemp bulls (n = 13) had an increased SST. After 60 min, LowTemp bulls had higher T concentrations compared to HighTemp bulls: 14.32 ng/ml ± 0.53 vs 10.30 ± 1.37 ng/ml (mean ± SEM; p
- Effect of Artificial Insemination Protocol and Dose of Frozen/Thawed
Stallion Semen on Pregnancy Results in Mares
- Authors: JLJ Govaere; MK Hoogewijs, C De Schauwer, S De Vliegher, A Van Soom, L Duchateau, A Kruif
First page: 487
Abstract: Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen–thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi-flexible Minitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p = 0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p = 0.0123).
- Detection of the Matrix Metalloproteinases MMP-2 and MMP-9 and Tissue
Inhibitors of Metalloproteinases TIMP-1 and TIMP-2 in Llama (Lama glama)
- Authors: R Zampini; ME Argañaraz, DC Miceli, SA Apichela
First page: 492
Abstract: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte–spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP-2, MMP-9, TIMP-1 and TIMP-2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP-2 and pro-MMP-9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero-tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.
- Efficiency of Oestrous Synchronization by GnRH, Prostaglandins and
Socio-Sexual Cues in the North African Maure Goats
- Authors: M Rekik; H Ben Othmane, N Lassoued, C Sakly
First page: 499
Abstract: This study aims to develop at different seasons, for local North African Maure goats, synchronizing protocols simultaneously to the standard ‘S’ protocol using progestagens in association with prostaglandins and gonadotropin. In late May, 40 goats were assigned to either the ‘S’ protocol or to a protocol where oestrus and ovulation were induced by the buck effect in single-injection progesterone-treated goats and provoking early luteolysis using prostaglandin 9 days after exposure to bucks ‘B’. During the 72 h after the treatments ended, 15 and 5 goats expressed oestrus in the ‘S’ and ‘B’ protocols (p 0.05). During mid-September, 60 goats were assigned to either ‘S’ treatment, ‘PGF’ treatment where oestrus and ovulation were synchronized using two injections of prostaglandin 11 days apart or to ‘GnRH’ treatment where the goats had their oestrus and ovulation synchronized with a GnRH (day 0)–prostaglandin (day 6)–GnRH (day 9) sequence. More ‘S’ goats were detected in oestrus over the 96-h period after the end of the treatments (88.8, 73.7 and 55% in ‘S’, ‘PGF’ and ‘GnRH’ treatments, respectively; p
- Direct Comparison of the Effects of Slow Freezing and Vitrification on
Late Blastocyst Gene Expression, Development, Implantation and Offspring
of Rabbit Morulae
- Authors: MD Saenz-de-Juano; F Marco-Jimenez, MP Viudes-de-Castro, R Lavara, JS Vicente
First page: 505
Abstract: This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.
- The Regulation of Aromatase and Androgen Receptor Expression During Gonad
Development in Male and Female European Eel
- Authors: DS Peñaranda; I Mazzeo, V Gallego, J Hildahl, R Nourizadeh-Lillabadi, L Pérez, F-A Weltzien, JF Asturiano
First page: 512
Abstract: This research investigated the regulation of aromatase and androgen receptor gene expression in the brain–pituitary–gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor α and β (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation.
- Immunohistochemical Localization of Fibroblast Growth Factor-2 in the
Sheep Ovary and its Effects on Pre-antral Follicle Apoptosis and
Development In Vitro
- Authors: JMS Santos; VG Menezes, RS Barberino, TJS Macedo, TLB Lins, BB Gouveia, VRP Barros, LP Santos, RJS Gonçalves, MHT Matos
First page: 522
Abstract: Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF-2 (p