Circulating levels of estradiol 17-β and progesterone vis à vis nitric oxide and nitric oxide synthase at and around oestrus in cycling buffaloes

Abstract

The present study was designed to measure the circulating levels of estradiol 17-β and progesterone vis à vis nitric oxide (NO) and nitric oxide synthase (NOS) levels at and around oestrus in cycling buffaloes. Blood samples were collected −2, −1, 0, +1 and +2 days of oestrus cycle from cycling buffaloes and analyzed for NO, NOS, estradiol 17-β and progesterone levels. In present study, the levels of NO did not show significant (P < 0.05) variation from −2 day to day of oestrus then decreased significantly and ranged between 11.4 and 26.4 μM/L. The NOS concentration showed similar pattern as exhibited by NO in buffaloes and ranged from 0.56 to 1.28 U/L. The estradiol 17-β in buffaloes gradually increased and peaked at day of oestrus and ranged between 1.9 and 29.32 pg/ml and the pattern was observed almost similar to those of NO and NOS. The levels of progesterone decreased from −2 day to day of oestrus and then increased and ranged between 0.01 and 0.42 ng/ml. The present study generates data of NO, NOS, estradiol 17-β and progesterone at and around oestrus in buffaloes and that is of practical importance for clinical and experimental interpretations.

Keywords:

• nitric oxide
• nitric oxide synthase
• estradiol 17-β
• progesterone
• oestrus
• buffalo

1. Introduction

Buffalo plays a prominent role in gross domestic product contribution from agriculture sector of India, by contributing about 50% of total milk production and more than 20% of the meat production (Uppal 2009). India produces about two-third of the world's buffalo milk and nearly half of the world's buffalo meat (FAOSTAT 2005). Problem of silent heat coupled with late maturity, poor expression of oestrus, irregular oestrous cycle, seasonality in breeding, anestrous, low conception rate, long postpartum interval, repeat breeding are some of the major constraints in buffalo productivity (Madan 1990). Besides the hormones, nitric oxide (NO) is also known to be an important regulator of the biology and physiology of the reproductive system. It is reported to have important role in regulation of steroidogenesis, follicle development, oocyte maturation, ovulation, luteal function and luteal regression (Jablonka-Shariff & Olson 1997; Basini & Tamanini 2000). It has been indicated that NO also plays a role in the optimal formation of corpus luteum in the early and middle periods of luteal phase, participates in the regulation of functional luteolysis at the end of the luteal phase (Skarzynski et al. 2000). Silent oestrus in buffalo is one of the major constraints in maximizing the dairy farm profitability and sustainability of milk production. The unobserved heat leads to suboptimal time of insemination resulting in prolonged calving interval (Lopez et al. 2004). Keeping in mind the important roles of gonadal hormones and NO in female reproductive cycle and for better understanding of the physiology of the reproductive cycle of buffaloes, the present study was designed to elucidate the roles of female hormones and nitric acid metabolism in reproductive cycle by estimating the circulating levels of NO (nitrite measurement), nitric oxide synthase (NOS), estradiol 17-β and progesterone at and around oestrus in cycling buffaloes.

2. Materials and methods

2.1. Experimental animals

Present study was conducted on eight normal cyclic Murrah buffaloes (aged 3–5 years) maintained at Instructional Livestock Farm Complex (ILFC) of College of Veterinary Science, Mathura. The animals were maintained at standard nutritional and managemental conditions throughout the study period. The animals utilized were regularly vaccinated against infectious and contagious diseases as per routine schedule.

2.2. Blood collection

Blood collection was started in the selected animals at the expected −5 days of oestrus cycle to +5 days of oestrus and day of oestrus was detected by teaser bull and rectal palpation. The blood samples of −2, −1, 0, +1 and +2 days of oestrus cycle were segregated and analyzed for biochemical parameters. Blood samples (15–20 ml approximately) were collected in dry, double-distilled water rinsed, heparinized screw capped vials through jugular venipuncture from selected animals following all hygienic measures. The samples were carried to laboratory in ice box and plasma was harvested by centrifugation at 3000 rpm for 15 min and stored at −20°C until analyzed for biochemical parameters.

2.3. Biochemical estimations

Blood samples were analyzed for NO and NOS using Enzchrom™ NOS assay kit, progesterone was estimated by liquid phase radioimmunoassay procedure using progesterone antisera raised in the Department of Veterinary Gynaecology and Obstetrics, GADVASU, Ludhiana and estradiol 17-β hormone was estimated using ELISA kits obtained from Diagnostics Biochem, Canada as per kit protocol.

2.4. Statistical analyses

Statistical significance was determined by an analysis of variance followed by Tukey's post hoc multiple comparison test using SPSS software for Windows (version 16.0). The data are presented as the mean ± SE. A p value < 0.05 was considered to be statistically significant.

3. Results and discussion

The circulating levels of NO (Nitrite measurement), NOS, estradiol 17-β and progesterone (mean ± SE) at and around oestrus in cycling buffaloes are depicted in Table 1. The results of present study showed highest concentration of NO and NOS during proestrus and oestrus (−2 to 0 day) which decreased significantly after day of oestrus. Similar trend of NO was also observed by Bulbul et al. (2008) in cattle and Rosselli et al. (1994) in buffaloes, corroborates the findings of present study. However, the values of NO reported in present study are slightly higher than the values reported by Bulbul et al. (2008) in cattle.

Table 1. Comparative profile of NO, NOS, estradiol 17-β and progesterone at and around oestrus in buffaloes.

	Days of oestrus cycle
Parameters	−2	−1	0	+1	+2
NO (μM/L)	22.25 ± 0.86^b	20.87 ± 0.83^b	20.12 ± 0.71^b	15.25 ± 0.25^a	13.75 ± 0.52^a
NOS (U/L)	1.10 ± 0.03^b	1.0 ± 0.05^b	1.0 ± 0.03^b	0.73 ± 0.01^a	0.66 ± 0.02^a
Estradiol 17-β (pg/ml)	19.78 ± 1.22^b	22.53 ± 1.03^bc	25.41 ± 0.97^c	3.21 ± 0.33^a	2.72 ± 0.32^a
Progesterone (ng/ml)	0.30 ± 0.23^c	0.19 ± 0.11^abc	0.09 ± 0.03^a	0.15 ± 0.02^a	0.27 ± 0.03^bc

Note: Values in the same row without a common superscript letter are significantly different (P < 0.05).

The estradiol 17-β concentration in present study is recorded comparable to those of Shafie et al. (1982) and Mondal et al. (2010), but lower than the values recorded by Bachalaus et al. (1979) and higher than the values observed by Bulbul et al. (2008) in buffaloes. The Table 1 showed increasing trend in levels of estradiol 17-β from −2 days to day of oestrus and then sudden fall significantly below the levels of days before oestrus. The pattern of estradiol 17-β concentration in oestrus cycle agrees with the earlier reports of other authors (Batra & Pandey 1983; Samad et al. 1988; Bulbul et al. 2008; Mondal et al. 2010).

The values of progesterone reported in present study is comparable to the results of Shafie et al. (1982) and Mondal and Prakash (2002), but observed to be lower than the values reported by Mondal et al. (2010) in buffaloes. The progesterone level showed decreasing trend from −2 days to day of oestrus and then gradually increased. The trend was observed to be similar to those of Mondal et al. (2007) and Mondal et al. (2010) confirm findings of present study. The trend of estradiol 17-β and progesterone showed normal physiological pattern of reproductive hormones of cycling animals.

NO synthesis depends on the action of NOS enzyme that exists in three iso-forms based on tissue of origin and on functional properties. Two of them, neuronal (nNOS) and endothelial (eNOS) are constitutive and seem to be responsible for the continuous basal release of NO; the third one is inducible (iNOS) and is expressed in response to inflammatory cytokines and lipopolysaccharides (Morris & Billiar 1994). In ovary, NO is synthesized by several ovarian cells such as theca, stroma, luteal and particularly granulosa cells in bovines (Basini et al. 1998; Basini et al. 2000) and both eNOS and iNOS observed to be involved, although their expression and activity greatly vary throughout different ovarian processes (Rosselli et al. 1998). The expression of both iNOS and eNOS is reported to be regulated by gonadotropins (Jablonka-Shariff & Olson 1997) as well as estradiol 17-β resulting in induction of NO synthesis in ovary (Al-Hijji et al. 2001; Dixit & Parvizi 2001).

Present study revealed sudden fall in levels of NO, NOS and estradiol 17-β following day of oestrus. NO and NOS exhibited a constant level up to day of oestrus then decreased following oestrus. Similarly estradiol 17-β revealed a steady but slow rise in concentration till oestrus and then exhibited a decline after oestrus. These trends of NO and estradiol 17-β are in agreement with the reports of Al-Hijji et al. (2001) and Dixit and Parvizi (2001). These trends might be due to the significant role of estradiol 17-β in generation of NO through induction of NOS activity. The concurrent rise in estradiol 17-β as well as in NO levels was also reported by Tamanini et al. (2003) and Bulbul et al. (2008) that indicates the role of estradiol 17-β in follicular development, steroidogenesis, ovulation and luteolysis may be through generation of NO in ovary (Tamanini et al. 2003). Increase in NOS activity in ovary corresponding to the time of the preovulatory surge suggests that NO may be assisting the process of ovulation via its stimulatory effect on prostaglandin production (Salvemini 1997), thus stimulating the inflammatory process at time of ovulation (Dixit & Parvizi 2001).

The steady concentrations of NO from +2 days up to oestrus followed by sudden fall observed in present study indicates that a certain level of NO is required to play a key role in luteolysis that may be due to oxytocin-mediated release of prostaglandin (Skarzynski et al. 2003). Oxytocin is reported to act by enhancing NOS activity (Motta & Gimeno 1997; Motta et al. 1997) and NO reported to stimulate the synthesis of PGF2α in human, (Friden et al. 2000), bovine (Skarzynski et al. 2000), which in turn increases NOS activity, thus activating a positive feedback mechanism (Motta et al. 2001). Simultaneously NO decreases progesterone production (Skarzynski & Okuda 2000). The role of NO in luteolysis has also been reported in human (Van Voorhis et al. 1994), rabbit (Gobbetti et al. 1999) and cow (Skarzynski & Okuda 2000). In a study in buffaloes, Sagar et al. (2012) reported that when NOS activity was inhibited by L-NAME, it resulted into increased progesterone production by corpus luteum. Similar kind of negative association was observed between NO and progesterone levels in present study, imitates the findings of present study.

4. Conclusions

The findings of present study provide the data on circulating levels of NO, NOS, estradiol 17-β and progesterone at and around oestrus in buffaloes that can be used for clinical and experimental interpretations. Regulation of oestrus cycle in buffaloes is very complex and involves a number of autocrine and paracrine mediators which exhibit a complex and intrinsic interplay of signal transduction mechanisms. These mechanisms are yet to be understood in buffaloes. Our study forms a basic layout in understanding the oestrus cycle in buffaloes and will further help in unravelling the molecular pathways involved in between them in the regulation of oestrus cycle. Further studies are required to elucidate the mechanisms by which NO influences the steroidogenesis, one of the most complicated phenomenon inside ovary in buffaloes and thus implicating the oestrus behaviour.

Acknowledgements

Authors are thankful to Vice Chancellor, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishvavidyalya evam Gau Anusandhan Sansthan, Mathura, for providing necessary facilities to carry out this work. Authors also thankful to Dr Rajneesh Sirohi and Dr D.N. Singh of Department of ILFC for providing help in management of animals and collection of blood samples.