Influence of climatic conditions on in vitro production of bovine embryos

Temperature and rainfall were analyzed daily during six years to evaluate their influence on in vitro production of bovine embryos. Weekly replications (n=480) were performed on 14,778 ovaries collected at slaughterhouses. Cumulus oocyte complexes (n=19,180) were fertilized with a pool of Bos taurus taurus semen in one incubator with 5% CO 2. Presumable zygotes were cultured in gasified plastic bags with 5% CO 2 , 5% O 2 , and 90% N 2 . In the first year, cleavage and embryo yield were 60.3% and 15.6%, respectively, being lower (P<0.05) than in the following years. Average cleavage rates were always lower in winter (P<0.0001), thus producing less embryos. Winter climatic conditions had a negative influence on in vitro production, when cleavage and embryo yield declined, possibly because of reduced availability and growth of native pasture.


INTRODUCTION
In vitro production (IVP) of bovine embryos comprises the steps of maturation (IVM), fertilization (IVF), and culture (IVC). This biotechnology has been used in Brazil since 1990(Oliveira et al., 1994 as a tool for the multiplication of animals with high genetic value and for cloning and transgenic animal production. Therefore, IVP is important in the scientific arena as well as in the industry. The native vegetation (Biome Pampa) of Rio Grande do Sul (RS) is also known as savannah, with the subtypes: shrubs and trees, and steppe (Quadros et al., 2003). The climate ensues a marked seasonality in the native pasture growth Arq. Bras. Med. Vet. Zootec., v.62, n.6, p.1381-1387, 2010 (Carvalho, 2009). The extensive management of animals on wide grazing areas reduces the productivity of the herd, in weight gain, and reproductive function. During the cold season in the southern hemisphere (May-September), the impairment on the growth of tropical forage plants is greater because of the occurrence of frost and irregular rainfall, resulting in a lower rate of forage accumulation, and consequently, lower daily average of body weight gain (Soares et al., 2005).
Environmental influences such as temperature, seasonality, and nutritional management interfere with reproductive efficiency, follicular development, and oocyte quality, and consequently, fertility (Webb, 1998;Armstrong et al., 2001;Webb et al., 2004). Environmental conditions and diets that are appropriate for follicular growth may not necessarily be appropriate for oocyte quality (Webb et al., 2004). Several studies demonstrate that nutritional and metabolic signals influence ovarian function by modulating the secretion of gonadotropins by the hypothalamic-pituitary axis (Gong, 2002). These observations focused on determining the nutritional influence on reproductive function mediated by the ovaries (Robinson et al., 2006).
The aim of this study was to analyze the potential influence of environment on bovine in vitro embryo production.
During all year around, for a period of 6 years, 480 replicates were performed to produce bovine embryos in vitro. Cumulus oocyte complexes (COC) were recovered from 14,778 ovaries of crossbred slaughtered cows (Bos taurus indicus x Bos taurus taurus) raised in Rio Grande do Sul, Brazil (latitude 29.00° to 31.00°S and longitude 52.00° to 54.00°W). The ovaries collected at slaughterhouses were kept in 0.9% saline solution at 30-35ºC during the transport of 22 to 92km to the laboratory.
The COC were recovered by aspiration of 2-8mm diameter follicles with an 18G needle connected to a vacuum pump (pressure 20mL/min). Cumulus oocyte complex selection was performed in follicular fluid (Lehmkul et al., 2000) under a stereomicroscope, and they were classified according to cumulus morphology (De Loos et al., 1989) into homogenous compact cytoplasm with unexpanded cumulus and expanded cumulus. The COC with unexpanded cumulus were randomly assigned to maturation and the expanded ones were discarded.
In vitro fertilization was performed with a pool of proved frozen semen from Bos taurus taurus bulls. After thawing semen in a water bath at 38°C for 30 seconds, the sperm was selected by swim up in Sperm-Talp medium (Parrish et al., 1995) with addition of 0.06mg/mL bovine serum albumin (BSA; A331) and 0.11mg/mL sodium pyruvate. After maturation, oocytes were transfered to four-well Nunc dishes containing 400μL Fert-Talp with 0.06mg/mL bovine serum albumin (BSA) and 0.022mg/mL sodium pyruvate supplemented with 50μg/mL heparin (H3393). Oocytes were inseminated with 1 to 2x10 6 sperm/mL and maintained at 38°C with 5% CO 2 in saturated humidity for 18 to 22 h. Zygotes were vortexed for 90 seconds and washed in 100μL drops of TCM-HEPES. Then, they were transfered for culture to 400μL SOFaaci medium (Holm et al., 1999) supplemented with 5% EMS and 0.022mg/mL sodium pyruvate, covered with 300μL mineral oil (M5904), previously stabilized in the incubator at 38°C with 5% CO 2 and saturated humidity. On day 2 (D2) of culture (D0 = fertilization day), the cleavage rate was assessed by the identification of cell division. Embryo culture was performed for four days in a foil bag system similar to the culture system described by Vajta et al. (1997) under an atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 . On day 7 (D7) of culture, embryos were evaluated and classified according to quality and development stage following IETS criteria.
The influence of temperature and rainfall was evaluated by correlation and regression analysis. Cleavage and blastocyst yield were analyzed by variance analysis. Graphics were produced by GraphPad Prism 5. Data were processed using SAS 9.1 statistical program. Differences were considered significant at P<0.05.

RESULTS
The climate in the central region of the RS state is characterized by average monthly temperatures below 17ºC from middle Autumn to the end of Winter (May-September) with frequent occurrence of frost in July and August. The average monthly temperatures from spring to summer (October-March) stay above 19ºC ( Figure 1A).
Cleavage ( Figure 2A) and the IVP rates on D7 ( Figure 2B) of the four seasons during six years highlights a reduction during the winter months.

DISCUSSION
The average annual precipitation volume was one factor that might have influenced the growth of natural and cultivated pastures. Flooding of the soil by intense rain can lead to reduction in the rate of carbohydrates, translocation from leaves to roots, reduced growth and metabolic activity of roots (Dias-Filho, 2005) and, consequently, lower nutritional quality of pasture.
The decreased accumulation of forage and increased amount of fibers found in pasture from autumn to winter (April-September) is mainly due to the low temperatures, luminosity, and frost characteristic of the climate. This reflects the seasonal production of native pasture in the region (Soares et al., 2005). During this study period, the lowest rainfall volume occurred from summer to late winter (January-August). This reduces the growth rate of native forage and changes the bromatological characteristics (Castro et al., 1997). . Med. Vet. Zootec., v.62, n.6, p.1381-1387 The negative impact on animal weight gain occur essentially during the winter months (June to August) due to the reduced availability and decreased growth of native pasture consequent to the low environmental temperature (Soares et al., 2006;Carvalho, 2009).

Arq. Bras
The weight loss in cows impairs the hormonal production at the hypothalamic-pituitary axis, the ovarian follicular growth (Adamiak et al., 2005), and oocyte quality (Lequarre et al., 2005). The most efficient test to assess oocyte quality is their ability to be fertilized, and to develop into the blastocyst stage (Lonergan et al., 2001). Thus, the most reliable parameter for oocyte quality evaluation is its competence to develop in vitro, and reach the first cleavage (Vam Soom et al., 1992). This finding is supported by the research of Van Soom et al. (1992) showing that oocyte quality and subsequent cleavage is influenced by the metabolic conditions of the donor. The cows whose ovaries were used in this study were grazing on native pastures during winter. The low availability of forage during this season results in gradual weight loss (Carvalho, 2009).
Weight loss negatively affects growth rate and ovulatory follicle size, resulting in changes in oocyte competence and development by low plasmatic glucose concentrations (O'Callaghan and Boland, 1999;Adamiak et al., 2005). Glucose is the primary metabolic fuel used by the central nervous system, and inadequate availability of glucose reduces hypothalamic release of GnRH (Keisler and Lucy, 1996;Wettemann et al., 2003). The ability of beef cows to maintain fairly constant concentrations of blood glucose, however, has prompted some reviewers to suggest that the role of glucose in mediating nutritional control of reproduction is permissive rather than causative (Schillo, 1992;Keisler and Lucy, 1996). This effect is probably due to the biological ability to store nutrients during the seasons of higher pasture availability for maintenance of physiological activity under adverse conditions (Armstrong e Evans, 1983). Low blood glucose may be detected by the hypothalamus in a threshold-dependent manner such that GnRH secretion will be impaired if glucose availability is inadequate (Randel, 1990;Dhuyvetter and Caton, 1996).
Thus, the reduction in cleavage and blastocyst yield during the cold season (July-August) may be an effect of low availability of native pasture growth during winter. Decrease in the cleavage rate from 79% to 64% was observed in sheep subjected to in vitro production with food restriction (O'Callaghan and Boland, 1999;Borowczyk et al., 2006). This finding corroborates with studies in cattle, in which ovaries were obtained from slaughtered cows under feed restriction during the cold season. Cows with low body score due to food restriction have lower levels of insulin growth factor-1 (IGF-I) and lower number of follicles larger than 8mm, compared to cows not exposed to food restriction (Boland et al., 2001;Gong, 2002;Adamiak et al., 2005).
Low IGF-1 levels increase the responsiveness of the ovarian follicle to FSH (Armstrong et al., 2001). In contrast, high dietary carbohydrate intake increases the concentration of circulating insulin and IGF-1, improving rate the growth of the dominant follicle.In females with decreased glucose levels, oocyte quality is directly affected and this is reflected by lower cleavage rates (Sutton et al., 2003). The glucose in oocytes is predominantly metabolized via the pentose phosphatase pathway for synthesis of DNA and RNA (Sutton-McDowall et al., 2004). Thus, maternal mRNA and protein molecules are synthesized and accumulated during oocyte growth and maturation, which are essential to ensure the embryo survival until the stage of 8-16 cells (Lonergan et al., 2003).

CONCLUSIONS
Extragonadal factors such as seasonal climatic changes, food intake, and nutritional status of the animals are correlated with IVP of bovine embryos. The decline in food supply available during the winter season leads to weight loss and impairs endocrine reproductive function. This correlation between climatic changes and reproductive parameters has been shown evaluating cleavage and blastocyst yield of in vitro produced bovine oocytes during the months of lower availability of native pasture.