Ovarian follicular synchronization, ovulation and oocyte development in llamas and alpacas

The purpose of the studies reported in this thesis was to increase our understanding of the reproductive physiology of South American camelids. Studies were conducted in llamas and alpacas to investigate methods to electively control ovarian follicular dynamics, to determine the effects of hormone preparations or biological factors derived from seminal plasma on ovulation induction, and to evaluate the establishment of superstimulatory protocols to induce a consistent ovarian follicular response for oocyte collection. The first study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas, and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating. In the first experiment, lutenizing hormone (LH) and follicular ablation treatments were most effective for inducing follicular wave synchronization, while estradiol plus progesterone (E/P) treatment was intermediate. In the second experiment, llamas were assigned randomly to Control, (E/P), and LH groups. A single, fixed-time natural mating was permitted 10 to 12 days after treatment. The pregnancy rate was higher (

Luteotrophic effect of OIF/NGF in llamas

An Ovulation-Inducing Factor (OIF) isolated from llama seminal plasma has recently been identified as Nerve Growth Factor (NGF). This protein not only has a potent ovulatory but also a luteotrophic effect in llamas after intramuscular or intrauterine infusion. The objective of the present review is to summarize the results of the effect of OIF/NGF on Corpus Luteum (CL) development in llamas. The results of a series of experiments demonstrate that the luteotrophic effect of OIF/NGF is mediated by a change in the secretion pattern of the LH surge. The intramuscular administration of OIF/NGF increases progesterone secretion regardless of the size of the preovulatory follicle at the time of treatment. The repeated administration of OIF/NGF during the periovulatory period (preovulatory, ovulatory, early postovulatory) increases CL size, vascularization and progesterone production. The effect of OIF/NGF varies with the route of administration (intramuscular, intravenous and intrauterine). It was concluded that OIF-NGF elicits a preovulatory LH surge followed by ovulation and development of a functional CL, regardless of the route of administration. However, with the intrauterine treatment, a physiological dose of OIF/NGF was required to elicit pituitary and ovarian responses

Ovulation-inducing factor: a protein component of llama seminal plasma

<p>Abstract</p> <p>Background</p> <p>Previously, we documented the presence of ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s) in seminal plasma responsible for inducing ovulation.</p> <p>Methods</p> <p>In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group) were treated i.m. with whole seminal plasma (positive control), phosphate-buffered saline (negative control), or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or < 5 kDa. In Experiment 2, female llamas (n = 7 per group) were given an i.m. dose of seminal plasma treated previously by: 1) enzymatic digestion with proteinase-K, 2) incubation with charcoal-dextran, 3) heating to 65°C, or 4) untreated (control). In Experiment 3, female llamas (n = 10 per group) were given an i.m. dose of pronase-treated or non-treated (control) seminal plasma. In all experiments, llamas were examined by transrectal ultrasonography to detect ovulation and CL formation. Ovulation rate was compared among groups by Fisher's exact test and follicle and CL diameters were compared among groups by analyses of variance or student's t-tests.</p> <p>Results</p> <p>In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each), but none ovulated in the other groups (P < 0.001). In Experiment 2, ovulations were detected in all llamas in each treatment group; i.e., respective treatments of seminal plasma failed to inactivate the ovulation-inducing factor. In Experiment 3, ovulations were detected in 0/10 llamas given pronase-treated seminal plasma and in 9/10 controls (P < 0.01).</p> <p>Conclusions</p> <p>We conclude that ovulation-inducing factor (OIF) in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.</p

Desarrollo de embriones de bovino obtenidos por fecundación in vitro cultivados con células oviductales o medio condicionado y transferidos a hembras receptoras

Se comparó el desarrollo in vitro de ovocitos obtenidos de ovarios de vaca de matadero, madurados, fecundados y cultivados in vitro bajo dos sistemas. Los ovocitos fueron cultivados en un medio de maduración a 39 &deg;C, 5 % de CO2 y humedad relativa de 95 % durante 22 horas. Posteriormente, fueron incubados con espermatozoides seleccionados a través de una gradiente discontinua de Percoll. La tasa de maduración nuclear y fecundación fueron de 93,7 % (74/79) y 76,9 % (50/65) respectivamente. Un total de 252 ovocitos fecundados fueron cultivados in vitro. El porcentaje de desarrollo in vitro a las 2 días post-inseminación (embriones de 4-8 células) fue de 62,7 % (64/102) para los cigotos cultivados con células oviductales y de 67 % (100/150) para los cultivados en medio condicionado (P0,05). El porcentaje de desarrollo de mórulas fue de 17,6 % (18/102) para los cigotos cultivados con células oviductales y de 13,3 % (20/150) para los cultivados con medio condicionado (P0,05). Se obtuvo una tasa de desarrollo del 15,7 % (16/102) de blastocistos para aquellos cigotos cultivados con células oviductales. No se obtuvo blastocistos a partir de cigotos cultivados en medio condicionado. Cuatro blastocistos fueron transferidos a dos hembras receptoras. A los 42 y 57 días se encontró la presencia de un feto en cada hembraThe in vitro development of matured and fertilized bovine oocytes was compared between two culture systems. Oocytes were collected by aspiration of follicles of 3-8 mm in diameter using an 18g needle. After morphological selection the oocytes were incubated at 39 0C, 5 % C02 y 95 % relative humidity, during 22 hours. Afterwards, oocytes were incubated with spermatozoa selected by Percoll gradient system. The rate of nuclear maturation and fertilization was 93,7 % (74/79) and 76,9 % (50/65), respectively. A total of 252 zygotes were cultured, 102 with oviductal cells and 150 in conditioned medium. The in vitro development on day 2 of culture (4- or 8-cell embryos) was 62,7 % (64/102) for the zygotes co-cultured with oviductal cells and 66,7 % (100/150) for the zygotes cultured in conditioned medium. The development to the morula stage was 17,6 % (18/102) for the zygotes co-cultured with oviductal cells and 13,3 % (20/150) for the zygotes cultured in conditioned medium. A statiscally significant difference was not found in the development of 4, 8-cell embryos or morula. The development of embryos up to the blastocyst stage was 15,7 % (16/102) for the zygotes co-cultured with oviductal cells. Two blastocysts were transferred to the uterine horn ipsilateral to the CL in two recipients by non-surgical embryo transfer. Pregnancy was confirmed by ultrasonography at 42 and 57 days detecting the presence of one conceptus in each animal. This work has shown that in vitro inseminated of bovine oocytes with espermatozoa prepared with modified BO and co-cultured with oviductal cells, can develop to the blastocysts stage, unlike those that were cultured with conditioned medium. Finally, it is important to mention that this is the first communication in Chile of pregnancy after in vitro fertilization of bovine oocyte

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n = 7 per group) were treated with: (1) 200 mg of FSH im divided bid for 3 d, plus a single i.v. dose of 1000 IU hCG 24 h after the last FSH treatment, or (2) 1200 IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000 IU of hCG on day 3 after eCG treatment (day 0 = start of superstimulatory treatment). At 20-24 h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles ≥6 mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles ≥6 mm at the time of COC collection (20.0 ± 7.5 versus 27.0 ± 3.3; P = 0.5), the number of COC collected (26.2 ± 8.4 versus 23.3 ± 3.7; P = 0.7), or the collection rate per follicle aspirated (89% versus 87%; P = 0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5 ± 2.9 versus 8.8 ± 2.8; P = 0.54), the number of expanded COC in metaphase II (8.5 ± 1.9 versus 6.0 ± 2.1; P = 0.1), or the number of compact COC with ≥3 layers of cumulus cells (12.5 ± 4.3 versus 14.3 ± 2.6; P = 0.72). A greater proportion (P < 0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18 h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26 h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro

Local versus systemic effect of ovulation-inducing factor in the seminal plasma of alpacas

BACKGROUND: Camelids are induced (reflex) ovulators. We have recently documented the presence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. The objective was to test the hypothesis that OIF exerts its effect via a systemic rather than a local route and that endometrial curettage will enhance the ovulatory response to intrauterine deposition of seminal plasma in alpacas. METHODS: Female alpacas were assigned randomly to 6 groups (n = 15 to 17 per group) in a 2 × 3 factorial design to test the effect of seminal plasma versus phosphate-buffered saline (PBS) given by intramuscular injection, by intrauterine infusion, or by intrauterine infusion after endometrial curettage. Specifically, alpacas in the respective groups were given 1) 2 ml of alpaca seminal plasma intramuscularly, 2) 2 ml of PBS intramuscularly (negative control group), 3) 2 ml of alpaca seminal plasma by intrauterine infusion, 4) 2 ml of PBS by intrauterine infusion (negative control group), 5) 2 ml of alpaca seminal plasma by intrauterine infusion after endometrial curettage, or 6) 2 ml of PBS by intrauterine infusion after endometrial curettage (negative control group). The alpacas were examined by transrectal ultrasonography to detect ovulation and measure follicular and luteal diameters. RESULTS: Intramuscular administration of seminal plasma resulted in a higher ovulation rate than intrauterine administration of seminal plasma (93% versus 41%; P < 0.01), while intrauterine seminal plasma after endometrial curettage was intermediate (67%). None of the saline-treated controls ovulated. The diameter of the CL after treatment-induced ovulation was not affected by the route of administration of seminal plasma. CONCLUSION: We conclude that 1) OIF in seminal plasma effects ovulation via a systemic rather than a local route, 2) disruption of the endometrial mucosa by curettage facilitated the absorption of OIF and increased the ovulatory effect of seminal plasma, and 3) ovulation in alpacas is not associated with a physical stimulation of the genital tract, and 4) the alpaca represents an excellent biological model to evaluate the bioactivity of OIF

Evidencia serológica de infección por Neospora caninum en rebaños lecheros del Sur de Chile

Neospora caninum is a recently recognised parasite that produces infection in dogs and herbivorous. In cows, the disease does not produce clinical signs of illness, instead it produces losses. Chile has a large dairy industry and pregnancy losses in that system seem to be economically important. However a large number of abortions remain with unknown aetiology and abortion due to protozoa has not been investigated. We report here, for the first time, that aborted cows belonging to three different farms in the IX Region, Chile had antibodies to N. Caninumis given here. The episode of abortion was "sporadic" and occurred in adult cows. The sera were diluted in 1:200 and processed with the Neospora IFA test. Whole tachyzoite surface fluorescent was considered positive. Aantibodies were found in 10 of 21 adult cows, none of 2 heifers and 1 of 6 calves in farm A. In farm B, they were found in 7 of 24 adult cows and in 2 of 2 adult cows of farm C. All the aborted cows (2 on farm A; 3 aborted and 1 mummified on farm B; and 2 on farm C) were positive to the test. Taking into consideration the IFA test results and the negativity of serology to other common infections which cause bovine abortion, the hypothesis that these cows aborted due to Neospora infection is supported. How the disease has spread world wide is unknown, but most probably, international animal trade has contributed to help spread the disease. Before any recommendation can be made more extensive studies should be done in order to establish the prevalence and the economic impact of the disease. At the moment evidence that Neospora infection is already present in this country is presented. Therefore it should be included as a differential diagnosis of bovine abortion.Los abortos bovinos causan importantes pérdidas económicas a la ganadería mundial. Una parte importante de ellos permanecen aún con etiología desconocida, y de los diagnosticados, la mayoría corresponden a causas infecciosas. Dentro de ellos los abortos ocasionados por protozoos han tomado relevancia mundial. En Chile hasta ahora se desconocía su presencia. En este trabajo se presenta evidencia que vacas con antecedentes de abortos presentaron anticuerpos contra N. caninum detectados mediante la técnica IFA y se discute la posibilidad de que hayan ocurrido como consecuencia de la infección por este protozo

Beneficial Effect of Omega-3 Fatty Acids on Immune and Reproductive Endometrial Function

Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are known by their anti-inflammatory effects through mechanisms such as formation of specialized pro-resolving mediators (SPM), and more recently a new mechanism dependent on the free fatty acid (FFA) receptors has been studied. DHA and EPA have shown an effect on the release of prostaglandins (PGs) E2 and F2α in endometrial cells, two PGs that have key function in fertility. In addition, other molecules such as cyclooxygenase-2, IL-1β, NF-κB, and intracellular signaling pathways are also affected by omega-3 fatty acids in endometrial cells. In this chapter, we will expose the following issues: eicosanoids in fertility and immune function in the uterus, effect of omega-3 fatty acids on endometrial function: in vivo and in vitro studies, mechanisms of action of omega-3 fatty acids in endometrial cells, and perspectives in health and diseases

New Insights Into the Role of β-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (β-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the β-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of β-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, β-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The β-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. β-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. β-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and β-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of β-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.Fil: Barraza, Daniela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Sari, Luciana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Apichela, Silvana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucuman. Facultad de Agronomia y Zootecnia. Cátedra de Zootecnia General I; ArgentinaFil: Ratto, Marcelo Hector. Universidad Austral de Chile; ChileFil: Argañaraz, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentin