Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum

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Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum Benner G. Alves a,∗ , Kele A. Alves a , Aline C. Lúcio b , Muller C. Martins b , Thiago H. Silva b , Bruna G. Alves b , Lucas S. Braga b , Thiago V. Silva a , Marco A.O. Viu a , Marcelo E. Beletti b , José O. Jacomini b , Ricarda M. Santos b , Maria L. Gambarini a a Center for Studies and Research in Animal Reproductive Biology, College of Veterinary and Animal Science, Federal University of Goiás, Goiânia, GO, 74001-970, Brazil b Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Federal University of Uberlândia, Uberlândia, MG, 38400-902, Brazil

a r t i c l e

i n f o

Article history: Received 15 October 2013 Received in revised form 26 February 2014 Accepted 28 February 2014 Available online xxx

Keywords: Dairy cattle Electrolytes Follicular fluid Girolando Heat stress Oocyte

a b s t r a c t This study was designed to evaluate the influence of heat stress (HS) on the metabolic profile of serum and follicular fluid (FF), ovarian follicle development, and oocyte quality of Girolando dairy cows. Oocytes, blood, and FF (follicles ≥9 mm) samples were obtained at 30, 45, 60, 75, and 90 days postpartum in the summer and winter seasons. During transvaginal follicular aspiration, rectal temperature (RT), body condition score (BCS), number of ovarian follicles, and quality of oocytes were recorded. The ambient air temperature (AT) and relative humidity (RH) were also recorded to calculate the temperature humidity index (THI). Glucose, total cholesterol (TC), triglycerides (TG), urea, sodium (Na), potassium (K), and calcium (Ca) concentrations were determined using serum and FF samples. The RT, THI, and BCS loss were greater (P < 0.01) in the summer; however, glucose, Na, and K serum concentrations decreased in the same season (P < 0.05). Degenerated oocytes were positively associated (P < 0.05) with THI (r = 0.14) and AT (r = 0.13), and negatively associated with glucose (r = −0.12) and K (r = −0.11) serum concentrations. HS induces metabolic changes, which compromise the number of ovarian follicles and the follicular environment, thus resulting in morphologically damaged oocytes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction NOTE: NOTE: Over the last few decades, advances in the field of genetics associated with improved management have led to the selection of highly productive cows to meet the demand of the dairy industry. These improvements

∗ Corresponding author. Tel.: +55 34 3218 2248; fax: +55 34 3218 2494. E-mail address: [email protected] (B.G. Alves).

were generated as a consequence of the greater incidence of hormonal and metabolic disorders, which are detrimental to oocytes and embryos (e.g., reduced fertility). Although some studies have revealed a correlation between such disorders and high milk yield, others have indicated that a decrease in reproductive performance is associated with a number of factors, including reproductive diseases, heat stress (HS), and weight loss (Bilby et al., 2006; Sewalem et al., 2008; Shehab-El-Deen et al., 2010).

http://dx.doi.org/10.1016/j.anireprosci.2014.02.019 0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.019

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HS is one of the most important factors responsible for decreased fertility in dairy cattle (Dobson and Smith, 2000). It is estimated that ∼60% of dairy cattle are affected by high temperatures during the summer (Roth et al., 2001), especially in the tropical and subtropical regions. HS impairs the estrous cycle of cows, thus resulting in a delay effect, attenuated follicular dominance, and a decrease in follicular steroid production (Wolfenson et al., 1997; Bilego et al., 2013). In Brazil, the majority of dairy production systems utilize the Girolando breed of cows because they combine the high productivity of Holstein cattle (Bos taurus) with the rusticity and thermal adaptations of Gir (Bos indicus) (Alves et al., 2013). The management of Girolando herds is conducted in the tropical savannah region in Brazil (i.e., the Brazilian Cerrado biome), which is characterized by a hot, semi-humid, seasonal climate with rainy summers and dry winters. Metabolic changes that are induced by lactation and associated with HS might impair ovarian function and the environment where the oocyte is located, thus reducing oocyte competence. Therefore, because of the importance and relationship between these factors and fertility, this study was conducted to evaluate the effects of HS on the serum metabolic profile, ovarian activity, oocyte quality, and follicular fluid (FF) metabolic status from dairy cows during the early lactation period. 2. Materials and methods 2.1. Animals and location Girolando breed cows (B. taurus × B. indicus) from the third lactation were assessed during the winter (n = 30) and summer (n = 30) seasons between July 2011 and February 2012. The experiment was conducted at the dairy research unit of the Federal University of Uberlândia, located in the tropical Cerrado biome (classified as the Cwa type according to Köppen), 18◦ 55 08 S latitude and 48◦ 16 37 W longitude, 776 m above sea level (Rubel and Kottek, 2010). The cows were milked twice a day, and the mean production of the herd per lactation period (305 days) was 5947.5 kg. During the summer, the animals were fed Cynodon spp. cv. Tifton 85 (ad libitum), supplemented with ration and minerals [bromatologic composition of total diet in the summer was as follows: % dry matter (%DM) = 15.1% crude protein (CP); 62.0% total digestible nutrients (TDN); 23.7% acid detergent fiber (ADF); 41.5% neutral detergent fiber (NDF); 2.6% ether extract (EE); 1.3% calcium (Ca); 0.6% phosphorus (P); 2.3 Mcal/kg DM of metabolizable energy (ME); and 1.4 Mcal/kg DM of liquid energy (LE)]. During the winter, the cows were confined to feedlots with a ration, sorghum silage, and minerals (bromatologic composition of total diet in the winter: %DM = 13.9% CP; 65.0% TDN; 26.2% ADF; 40.7% NDF; 2.2% EE; 1.2% Ca; 0.4% P; 2.4 Mcal/kg DM ME; and 1.5 Mcal/kg DM LE). The diets were formulated according to production nutritional requirements (NRC, 2001). 2.2. Rectal temperature (RT), body condition score (BCS), and weather variables The BCS was scored based on a scale of 1–5, with 0.25 increments (Edmonson et al., 1989), and the RT

was measured prior to follicular aspiration using a digital thermometer inserted into the rectal area. Environmental variables were obtained from the weather station of the Climatology Laboratory from Uberlândia Federal University. The ambient air temperature (AT, ◦ C) and relative humidity (RH, %) were used to calculate the temperature humidity index (THI) according to Mader et al., 2006: THI = (0.8 × AT) +

 RH  100



× (AT − 14.4) + 46.4

2.3. Transvaginal follicular aspiration (TFA)–FF and oocyte collection We conducted 300 TFA procedures (n = 150, summer; n = 150, winter) throughout the study. Each cow (n = 30, summer; n = 30, winter) was subjected to the TFA procedure at 30, 45, 60, 75, and 90 days after parturition. These procedures were started, on average, at 31.2 ± 1.2 and 32.5 ± 2.4 days postpartum in the summer and winter seasons, respectively. The TFA was performed using ultrasound scanner (SSD-500; Aloka, Tokyo, Japan), with a 5-mHz micro convex transducer connected to a biopsy guide (WTA; Watanabe Applied Technology, Cravinhos, Brazil) and a suction line (WTA) with a 16G × 5.2 inch catheter (1.7 × 1.3 mm; BD, Curitiba, Brazil). Caudal epidural anesthesia was induced with 4 mL of 2% lidocaine (Eurofarma, São Paulo, Brazil), the perineum area was scrubbed, and the aspiration guide with the micro convex transducer was inserted; the size and number of the follicles from the ovaries were classified as small (2–4 mm), medium (5–8 mm), or large (≥9 mm). The FF was aspirated from the large follicles (when present) using a 5-mL syringe (Descarpack, São Paulo, Brazil) coupled to the aspiration system. Immediately after collection, the samples were centrifuged (2100 × g, 30 min, 25 ◦ C), and the supernatant was stored at −20 ◦ C until analysis. Only the FF samples that were not contaminated with blood were analyzed. Oocytes were obtained from follicles (2–8 mm in diameter) using constant vacuum pressure of the equipment, which was adjusted to a volume of water per minute (16 mL/min) in a medium with phosphate buffered saline (PBS), with additions of 10% of fetal bovine serum (FBS; Cultilab, Campinas, Brazil) and 7.5 UI/mL of sodium heparin (Liquemine; Roche, São Paulo, Brazil). The solution was filtered to hold the oocytes, which were transferred to Petri dishes (60 × 15 mm, Cultilab) containing a tissue culture medium (TCM-199; Sigma, St. Louis, USA) supplemented with HEPES (20 mM; Sigma), sodium bicarbonate (5 mM; Sigma), pyruvate (4 mM; Sigma), FBS (10%; Cultilab), and amikacin sulfate (80 ␮g/mL; Sigma). 2.4. Oocyte classification The cumulus-oocyte complexes (COC) were scored for quality with the aid of a stereoscope (×40), as described by Walters et al., 2002, as follows: Score 1 (GI), more than three compact layers of cumulus-oocyte cells and a homogeneous cytoplasm; Score 2 (GII), two compact layers of cumulus-oocyte cells and a less homogeneous than GI; Score 3 (GIII), irregular layer with a few cumulus-oocyte

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cells and dark agglomerations in the cytoplasm; and degenerated oocyte (DEG), absence or spread of cumulus-oocyte cells, and irregular and dark cytoplasm agglomerations. For the present study, oocytes GI, GII, and GIII were considered viable. 2.5. Blood samples Blood samples were simultaneously obtained alongside the TFA from all animals utilized throughout the experiment during the summer and winter seasons. The coccidian vein was punctured to draw blood through an 18 G × 1.5 inch hypodermic needle (BD, Curitiba, Brazil) in sterile vacuum tubes. The samples were kept inside the isothermal box (4 ◦ C) until transported to the laboratory where they were centrifuged (2100×g, 30 min, 25 ◦ C). The serum was stored in aliquots at −20 ◦ C until use in the biochemical analysis.

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Table 1 Mean ± (SEM) and variation of ambient air temperature (AT), relative humidity (RH), and temperature humidity index (THI) during summer and winter. Season

AT (◦ C)

Summer

23.8 ± 0.1 (18.6–32.5) 22.9 ± 0.1 (13.5–33.2)

Winter a

RH (%) a

THI

66.4 ± 1.5 (31.0–95.0) 40.5 ± 0.7 (14.0–74.0) a

71.0 ± 0.04a (69.2–71.5) 67.9 ± 0.1 (64.9–70.1)

Differences between seasons (P < 0.01).

3. Results

2.7. Statistical analysis

The results are reported as the least square means and standard error of the mean (mean ± SEM). It was not possible to obtain FF with 11% of the TFA procedures due the absence of a large follicle or blood contamination. The AT, RH, THI, and RT (38.5 compared with 38.2 ◦ C) were greater (P < 0.01) for the summer when compared to the winter, thus characterizing the occurrence of HS (Table 1). BCS loss occurred in both seasons between 30 and 90 days postpartum (P < 0.05). However, during the summer, there was a greater loss of points on the BCS scale when compared to the winter (0.89 ± 0.04 compared with 0.39 ± 0.32; P < 0.05) up until 90 days of lactation. Further, cows from the summer group exhibited no significant difference between 30 (2.4 ± 0.1) and 90 days (2.6 ± 0.08) following parturition. The BCS for the winter group was greater on day 90 (3.0 ± 0.05 compared with 2.6 ± 0.08; P < 0.01; Fig. 1). In the experiment, 3805 ovarian follicles were counted prior to the TFA procedures. The mean number of follicles recorded for each cow regardless of the size was greater (P < 0.05) during the winter (13.7 ± 0.4 compared with 11.6 ± 0.6), mainly on days 30 (14.4 ± 0.7 compared with 9.2 ± 0.5; P < 0.01) and 90 (14.4 ± 0.9 compared with 11.3 ± 0.9; P < 0.05) of the lactation period (Fig. 2a). Cows showed a greater number of small follicles (2–4 mm) during the winter when compared to the summer (8.0 ± 0.4 compared with 7.0 ± 0.6; P < 0.01), which was maintained until 90 days postpartum. During the summer, the lowest

The study followed a completely random design in a 2 × 5 factorial arrangement of treatments. Critical analysis and data consistency were performed using PROC UNIVARIATE (SAS Institute Inc., Cary, NC, USA), and data were transformed when necessary. Analyses of oocyte total number and viable oocytes, number of small (2–4 mm) and medium (5–8 mm) follicles, and oocyte quality (GI, GII, GIII, and DEG) were conducted using the logarithm transformation [log (X)]. For the number of large follicles (≥9 mm), the √ best fit was obtained through radix transformation ( x). Variance analyses were performed using the least-squares method using PROC GLM (SAS). Adjusted means and t tests were obtained using the LSMEANS option of PROC GLM (SAS). To study the association between two variables, the Pearson correlation coefficient was calculated using the CORR procedure (SAS). Correlation coefficients were classified as strong (r > 0.6), moderate (0.6 < r < 0.4), or weak (r < 0.4). The results were considered significantly different when P < 0.05.

Fig. 1. Mean ± (SEM) of body score condition (BSC) and rectal temperature (RT) of postpartum dairy cows in the summer and winter seasons. Differences between seasons for BCS (* P < 0.01) and RT (+ P < 0.01).

2.6. Biochemical analyses–Serum and FF Glucose concentrations were determined for each blood and FF sample during the TFA proceedings using test strips (Accu-Chek Active; Roche, São Paulo, Brazil). Serum analyses of total cholesterol (TC) and triglycerides (TG) were performed by an endpoint enzymatic reaction using commercial kits (Cholesterol Liquiform and Triglycerides Liquiform; Labtest Diagnostica, Lagoa Santa, Brazil). Urea was determined using an enzymatic reaction and ultraviolet photometry using the kinetic method (Urea UV Liquiform; Labtest Diagnostica). All analyses were performed using the semiautomatic biochemical equipment Bio-2000 BIOPLUS (Bioplus Laboratory Products Ltd., São Paulo, Brazil). The dosage of the sodium (Na), potassium (K), and calcium (Ca) ions were determined by the ion-selective electrode method using the AVL 9180 electrolytic analyzer (Roche, São Paulo, Brazil). All procedures were performed according to the manufacturer’s instructions, and the intraand inter-assay coefficients of variation were <5%.

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.019

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Fig. 2. Mean ± (SEM) of ovarian follicles recorded before transvaginal follicular aspiration, between 30 and 90 days postpartum, during the summer and winter seasons. (a) Total ovarian follicles, (b) small follicles (2–4 mm), (c) medium follicles (5–8 mm), and (d) large follicles (≥9 mm). Differences between seasons (* P < 0.05; + P < 0.01).

number of small follicles was observed at 30 days postpartum (4.3 ± 0.5; P < 0.05; Fig. 2b). The number of medium follicles (5–8 mm) was greater (P < 0.05) during the winter (4.2 ± 0.2 compared with 3.5 ± 0.2); however, this number decreased (P < 0.05) during both seasons after 45 days of lactation. The number of follicles increased (P < 0.05) and there was a similar number of follicles at 60 days postpartum in the winter, which was not observed for the animals in the summer (i.e., recovery after 75 days of lactation; Fig. 2c). The number of large follicles (≥9 mm) was greater during the winter (1.5 ± 0.06 compared with 1.1 ± 0.07; P < 0.01); at 30 days postpartum, twice the number (P < 0.01) of large follicles (1.5 ± 0.1) was observed when compared to the summer (0.7 ± 0.1). Despite the greater amount of large follicles recorded during the winter, the difference was not significant (P > 0.05) at 45, 60, and 90 days of lactation (Fig. 2d). Following the TFA procedure, 1936 oocytes (recovered index: 50.8%) were obtained. The number of oocytes per cow did not differ between the seasons (summer: 7.0 ± 0.8 compared with winter: 5.9 ± 0.3; P > 0.05), and the comparison of the means of oocytes between days within the

season did not show any difference (P > 0.05) between the summer (day 30: 4.7 ± 0.4 compared with day 90: 5.3 ± 0.5) and winter (day 30: 6.0 ± 0.6 compared with day 90: 6.6 ± 0.6) seasons (Fig. 3a). The means of viable oocytes did not differ between the seasons (summer: 3.16 ± 0.5 compared with winter: 3.14 ± 0.2). The number of viable oocytes was greater (P < 0.05) during the winter when compared to that in the summer at 30 (3.8 ± 0.4 compared with 1.7 ± 0.3), 60 (4.0 ± 0.9 compared with 2.2 ± 0.5), and 90 days (3.1 ± 0.5 compared with 1.6 ± 0.3) postpartum (Fig. 3b). The number of GI and GII oocytes did not differ between the summer and winter seasons (Fig. 4a, b). However, it was possible to recover more GIII oocytes in the winter when compared that in the summer (2.1 ± 0.2 compared with 1.9 ± 0.3; P < 0.05) (Fig. 4c). The mean of degenerated oocytes was higher during the summer (3.9 ± 0.3 compared with 2.7 ± 0.2; P < 0.01), with variation in oocyte quality observed throughout the study (Fig. 4d). The effect of season was demonstrated for the mean of GIII oocytes during the winter (P < 0.05) and degenerated oocytes in the summer (P < 0.01). The proportions of GI, GII, GIII, and DEG oocytes throughout the study were 4.3%, 11.4%, 28.3%, and

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Fig. 3. Mean ± (SEM) of total oocytes (a) and viable oocytes (b) recovered by transvaginal follicular aspiration in postpartum dairy cows during the summer and winter seasons. There were differences between seasons (* P < 0.05; + P < 0.01).

Fig. 4. Oocyte quality (mean ± SEM) of dairy cows between 30 and 90 days of lactation, during the summer and winter seasons. (a) Oocyte score 1 (GI), (b) oocyte score 2 (GII), (c) oocyte score 3 (GIII), and (d) degenerated oocytes (DEG). There were differences between seasons (* P < 0.05; + P < 0.01).

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(r = −0.11; P < 0.05) and a positive correlation with Ca (r = 0.17; P < 0.01) were also observed.

4. Discussion

Fig. 5. Oocyte quality distribution of dairy cows between 30 and 90 days of lactation in different seasons (summer and winter). GI: oocyte score 1; GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated oocytes. There were differences between seasons for oocyte quality (* P < 0.05; + P < 0.01).

56% for the summer and 6.8%, 10.1%, 35.6%, and 47.5% for the winter, respectively (Fig. 5). Metabolites and electrolytes in the serum (glucose, urea, TG, Na, K, and Ca) and FF (TC, TG, urea, K, and Ca) were influenced by seasonal variations (Table 2). Correlation coefficients from clinical and weather variables are shown in Table 3. The main results observed were a negative association (P < 0.01) between the total of ovarian follicles with RT (r = −0.14), RH (r = −0.18), and THI (r = −0.16); negative correlations between viable oocytes with THI (r = 0.19; P < 0.01) and AT (r = −0.13; P < 0.05); and positive correlations between degenerated oocytes with THI (r = 0.14; P < 0.01) and AT (r = 0.13; P < 0.05). Correlations between TC, urea, and Na, and the number of ovarian follicles, oocyte quantity, and oocyte quality were not observed (Table 4). Serum TG (r = 0.11) and K (r = 0.11) were, however, positively correlated (P < 0.05) with the number of ovarian follicles. Serum glucose concentration exhibited a positive correlation (P < 0.05) with the number of small (r = 0.12) and large follicles (r = 0.13), and a negative association (P < 0.05) with degenerated oocytes (r = −0.12). Furthermore, a negative correlation between the number of degenerated oocytes with K

The FF samples in the current study were collected every 15 days. Thus, the experimental design had some limitations. For example, some FF samples (≥9 mm) may have been collected from atretic follicles. Follicular deviation occurs when the follicle reaches an average of 8.5 mm in diameter (Ginther et al., 2001). However, it was decided in the present study to use all the samples due to the fact that the concentrations of glucose in the FF (mg/dL) during the summer (76.1 ± 2.8) and winter (77.1 ± 1.9) seasons were similar to concentrations described for active dominant follicles (Shehab-El-Deen et al., 2010; Moallem et al., 2011; Aller et al., 2013). In addition, some studies have shown that the stage of the estrous cycle does not affect the number of aspirated follicles or oocyte quality (Arlotto et al., 1996; Acar et al., 2013). Favorable conditions for HS development occurred during the summer in the present study, with greater weather variable values for AT, RH, and THI. Greater BCS loss and elevated RT occurred during the postpartum period in the summer. At the beginning of lactation, the energy requirement for maintenance and milk production is greater than the energy that cows gain from dry matter intake (DMI). These differences result in greater mobilization of the body tissue reserves, which is associated with a lesser DMI and a decrease in metabolic heat production due to an increase in body temperature imposed by THI that can contribute to reproductive dysfunctions (Dikmen and Hansen, 2009; Wolfenson et al., 2000). The effects of HS on follicles were evident during the summer because the number of follicles classified as small (2–4 mm), medium (5–8 mm), or large (≥9 mm) was greater during the winter. In general, negative correlations between the total number of follicles and RT, RH, and THI were identified. Changes in body temperature caused by HS during follicular development inhibited growth and ovulation due to a decrease in the receptors for LH, estradiol, and aromatase activity (Ozawa et al., 2005). Lactating dairy cows experiencing HS had a decrease in plasma

Table 2 Seasonal effect (summer and winter) of the metabolites concentration (mean ± SEM) on serum and follicular fluid (FF) of dairy cows between 30 and 90 days postpartum. Metabolites

Glucose (mg/dL) TC (mg/dL) TG (mg/dL) Urea (mg/dL) Na (mmol/L) K (mmol/L) Ca (mmol/L)

Follicular Fluida

Serum Summer

Winter

P-valueb

Summer

Winter

P-valuec

56.1 ± 0.7 136.9 ± 7.7 13.3 ± 0.7 23.6 ± 0.9 134.4 ± 1.5 4.3 ± 0.1 0.8 ± 0.01

63.5 ± 0.9 129.2 ± 4.2 11.3 ± 0.7 31.3 ± 1.5 139.2 ± 2.1 4.8 ± 0.1 0.6 ± 0.01

0.01 0.23 0.05 0.01 0.05 0.01 0.01

76.1 ± 2.8 83.3 ± 4.4 22.1 ± 1.9 13.3 ± 0.7 221.5 ± 9.8 4.7 ± 0.2 0.9 ± 0.1

77.1 ± 1.9 60.7 ± 3.1 14.3 ± 1.0 11.2 ± 0.6 206.6 ± 5.9 4.3 ± 0.1 0.5 ± 0.02

0.78 0.01 0.01 0.05 0.19 0.01 0.01

TC: total cholesterol; TG: triglycerides; Na: sodium; K: potassium; Ca: calcium. a Follicles ≥9 mm in diameter. b The P-values refer to the seasonal comparison of the average concentration of metabolites on serum. c The P-values refer to the seasonal comparison of the average concentration of metabolites on FF.

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Table 3 Correlation coefficients (r) between clinical and climatic variables with ovarian follicles, oocyte number, and oocyte quality from postpartum dairy cows. Variables

RT

BCS

THI

AT

RH

Total follicles Follicles 2–4 mm Follicles 5–8 mm Follicles ≥9 mm Total oocytes Viable oocytes Oocytes GI Oocytes GII Oocytes GIII Oocytes DEG

−0.14** −0.12* −0.04 −0.06 −0.05 −0.08 −0.06 −0.07 −0.05 0.00

0.05 0.01 0.01 0.00 0.01 0.08 0.03 0.04 0.11* −0.06

−0.16** −0.13** −0.13* −0.16** −0.05 −0.19** −0.06 −0.14** −0.19** 0.14**

0.07 0.02 0.09 −0.05 0.02 −0.13* −0.12* −0.11* −0.12* 0.13*

−0.18** −0.11* −0.18** −0.09 −0.01 0.02 0.02 0.01 0.03 0.01

RT: rectal temperature; BCS: body condition score; THI: temperature humidity index; AT: ambient air temperature; RH: relative humidity; GI: oocyte score 1; GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated oocytes. * Values are different (P < 0.05). ** Values are different (P < 0.01).

estradiol concentration and follicular diameter due to the lesser steroidogenesis in the granulosa and theca cells (Wilson et al., 1998). In addition, the difference in antral follicles recorded among the seasons could be due to individual cow variation (Burns et al., 2005). However, HS compromises the steroidogenic capacity of follicles, and dairy cows have fewer ovarian follicles (≥3 mm) which could be associated with reduced fertility (Mossa et al., 2012). Serum glucose concentration was less during the summer and was positively correlated with number of small (2–4 mm) and large (≥9 mm) follicles. Energy concentrations before and after parturition have a notable influence on the size and number of follicles. The growth of follicles was suppressed when a negative energy balance (NEB) was associated with BCS loss resulting in a decrease in the number of follicles (Perry et al., 1991). The decrease in energy concentrations consequently leads to a decrease in the concentration of IGF-1 and estrogen in the FF. The daily growth in size and total number of dominant follicles is influenced by the variability of energy intake, which is positive for cows fed diets with greater energy content (Kendrick et al., 1999). The average number of oocytes and the number of viable oocytes recovered in each TFA did not differ between the seasons; moreover, numbers were not influenced by postpartum duration. However, the average number of GIII oocytes was greater during the winter, and the number of

degenerated oocytes was greater in the summer. A negative correlation was observed between serum glucose concentration and the number of degenerated oocytes. The THI and AT variables had a negative association with the number of viable oocytes and a positive correlation with the number of degenerated oocytes. Furthermore, was observed a seasonal effect for metabolic and ion concentrations in serum (glucose, TG, urea, Na, K, and Ca) and FF (TC, TG, urea, K, and Ca). In a recent study, Matoba et al., 2012 indicated that lactation did not have an effect (up to 80 days postpartum) on the morphology or development of oocytes from dairy cows when were in vitro cultured. However, changes in the biochemical profile due to HS were reported for glucose concentration, IGF-1, NEFA, urea, and TC, which could compromise oocyte development and granulosa cell quality (Shehab-El-Deen et al., 2010). Serum metabolic TC, Na, and urea were not associated with the number of follicles or oocyte quality in the present study. The TG and K concentrations, however, were positively correlated with the number of follicles. The number of degenerated oocytes was negatively and positively correlated with K and Ca concentrations, respectively. Some studies reported correlations between concentrations of metabolic variables (e.g., glucose, TC, TG, and urea) and ions (e.g., sodium, chlorine, and calcium) in the serum and FF, which can change the microenvironment for oocyte development (Leroy et al., 2004; Alves et al., 2013). Studies

Table 4 Correlation coefficients (r) between serum metabolites with ovarian follicles, oocyte number, and oocyte quality from postpartum dairy cows. Variables Total follicles Follicles 2–4 mm Follicles 5–8 mm Follicles ≥9 mm Total oocytes Viable oocytes Oocytes GI Oocytes GII Oocytes GIII Oocytes DEG

Glucose 0.03 0.12* −0.04 0.13* 0.05 0.06 0.01 0.07 0.01 −0.12*

TC 0.03 0.02 0.06 0.01 0.00 −0.06 0.03 −0.01 −0.06 0.05

TG

Urea *

0.11 0.07 0.06 −0.01 0.02 0.03 −0.03 0.07 0.05 0.06

0.06 0.06 0.03 0.05 0.02 0.00 0.04 −0.05 0.00 −0.08

Na 0.05 0.00 0.09 0.00 −0.02 −0.02 −0.06 0.04 −0.01 −0.04

K

Ca *

0.11 0.04 0.18** −0.02 −0.03 0.00 0.00 0.04 0.00 −0.11*

0.04 0.05 −0.02 −0.08 0.01 0.00 −0.09 0.00 0.00 0.17**

TC: total cholesterol; TG: triglycerides; Na: sodium; K: potassium; Ca: calcium; GI: oocyte score 1; GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated oocytes. * Values are different (P < 0.05). ** Values are different (P < 0.01).

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.019

G Model ANIREP-4931; No. of Pages 9

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have already demonstrated that urea may impair meiosis, fertilization, in vitro embryo development (Sinclair et al., 2000; De Wit et al., 2001), and receptor expression for IGF1 and insulin in the endometrium during uterine involution (Wathes et al., 2011). However, there are other studies where the effects of urea on fertility were not observed in dairy cows (Oliveira et al., 2004; Rehak et al., 2009). Serum TG concentrations were positively correlated with the total number of follicles and was influenced by season; in addition, greater concentrations of TG occurred in the serum and FF during the summer. In addition to functioning as an energy source, lipids have an important role in the composition of membrane cells. The physical properties of the membranes are regulated by lipid constitution, environmental factors, and diet (Kim et al., 2001; Wonnacott et al., 2010). Zeron et al., 2001 observed inferior quality of cumulus oocyte complexes obtained during the summer with lesser cleavage indices and blastocyst development during in vitro production, and a greater percentage of saturated fatty acids in the oocytes, granulosa cells, and FF. However, in the present study there was not a relationship between TG concentrations and oocyte quality. Potassium concentration was greater in the FF during the summer and was negatively correlated with the number of degenerated oocytes. All electrolytes evaluated in the present experiment were affected by seasonal variation in both the FF and serum, except sodium, which was only affected in the serum. Sodium and potassium are constituents of sweat, and lesser serum concentrations of these elements could be found in the summer due to transpiration, which is the main thermoregulatory mechanism of zebuines (B. indicus; Kadzere et al., 2002). In maturation medium, different proportions of Na/K (16 and 24) were used (Iwata et al., 2004), and this variation in proportions did not influence oocyte competence or nuclear maturation in cattle. However, in mice, an increase in hatching rates of blastocysts in vitro was verified when the Na/K ratio was between three and 10 (Jin et al., 1994). In the present study, there was a ratio of 47 and 48 for Na/K in the FF during the summer and winter, respectively. However, the effect of this relationship on the reproductive performance of dairy cows is not clear. Serum Ca concentrations were positively correlated with the number of degenerated oocytes. Furthermore, serum and FF concentrations of Ca were greater during the summer. Ca has an important role in acquired meiotic competence and polyspermy block (He et al., 1997). Changes in Ca intracellular regulation occur during folliculogenesis (Rozinek et al., 2006) and oocyte maturation (Carroll et al., 1994). Lebedeva et al., 1998 identified the accumulation of intracellular Ca reserves in the granulosa cells of cattle from follicles undergoing atresia. Ca storage is associated with the morphologic quality of immature oocytes and the developmental potential of mature oocytes (Boni et al., 2002). In summary, HS modifies the serum profile and FF metabolic status in lactating dairy cows, thus affecting oocyte quality. Fluctuations in serum glucose, TG, K, and Ca are reflected in the follicular environment, thus resulting

in the production of morphologically damaged oocytes that can contribute to decreased fertility after parturition.

Conflict of interest None declared.

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Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.019