Attenuation of luteolytic response following fish meal supplementation in dairy buffaloes (Bubalus bubalis)

Animal Reproduction Science 126 (2011) 45–49

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Attenuation of luteolytic response following fish meal supplementation in dairy buffaloes (Bubalus bubalis) A.A. Malik a,∗ , V.K. Gandotra a , P.S. Brar a , S.P.S. Ghuman a , G.S. Dhaliwal b a b

Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004, India Department of Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004, India

a r t i c l e

i n f o

Article history: Received 29 September 2010 Received in revised form 13 April 2011 Accepted 20 April 2011 Available online 11 May 2011 Keywords: Antiluteolytic Buffalo Fish meal Oxytocin challenge PGFM

a b s t r a c t Luteolysis of corpus luteum, due to un-inhibited PGF2␣ secretion, has been reported to be a cause of early embryonic mortality in dairy animals. The objective of this study was to determine the effects of fish meal (FM) supplementation on the uterine secretion of PGF2␣ and hence establish its supplementation as an antiluteolytic strategy in dairy buffaloes. Five cycling Murrah buffaloes were supplemented with 250 g FM daily for 55 days in addition to their routine feed and seven buffaloes were kept as non-supplemented control. After 30 days of FM supplementation, the oestrus was synchronized in all the buffaloes using Ovsynch protocol. On day 15 of synchronized cycle, animals were challenged with oxytocin (OT; 100 IU) intravenously and blood samples were collected at 15 min interval, 1 h before to 4 h after OT challenge. The PGF2␣ response was measured as the venous concentration of 13,14-dihydro-15-keto PGF2␣ (PGFM). The mean hourly concentration of PGFM in FM supplemented buffaloes was lower than in the control buffaloes at all the occasions. During peak response (1 h post-OT challenge), PGFM concentration was significantly lower (P < 0.05) in FM supplemented buffaloes than in the control (197.4 ± 41.7 pg/ml versus 326.3 ± 33.5 pg/ml, respectively). Also the percent rise in PGFM after OT-challenge in FM supplemented buffaloes was less than the control (11.73% versus 22.47%). The dietary supplementation did not affect the size of corpus luteum (CL) and plasma progesterone concentration. Plasma glucose and total protein concentrations remained within the normal physiological limits during FM supplementation. The present study indicated that supplementing FM decreased the concentrations of PGF2␣ without alterations in the size of CL and plasma progesterone concentrations in dairy buffaloes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Division of Animal Reproduction, Gynaecology and Obstetrics, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir, University of Agricultural Sciences and Technology of Kashmir, Shuhama, Alusteng, Srinagar, Jammu and Kashmir 190006, India. Tel.: +91 9797134488; fax: +91 1942262208. E-mail addresses: [email protected] (A.A. Malik), [email protected] (V.K. Gandotra), [email protected] (P.S. Brar), ghuman [email protected] (S.P.S. Ghuman), [email protected] (G.S. Dhaliwal). 0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2011.04.010

Repeat breeding is becoming a major reproductive constraint in dairy buffalo rearing (Sah and Nakao, 2006). In cattle, early embryonic mortality has been recognized as the major contributor to this disorder, while similar studies in dairy buffaloes are lacking. Majority of embryonic loss (30%) occurs between days 8 and 16 post-insemination in dairy cattle (Diskin and Sreenan, 1980; Sreenan et al., 2001). Embryo loss at this stage may be associated with an inability to inhibit the luteolytic action of prostaglandin F2␣ (PGF2␣ ) during the critical period of maternal recognition of pregnancy. In the pregnant cow interferon-tau (IFN-␶)

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A.A. Malik et al. / Animal Reproduction Science 126 (2011) 45–49

produced by the conceptus between days 14 and 18 of pregnancy inhibits uterine produced PGF2␣ , thus facilitating progesterone mediated maintenance of pregnancy (Burns et al., 2003). However, ability of conceptus to synthesize IFN-␶ depends upon its size (Binelli et al., 2001) which is highly variable (15–250 mm) on day 17 of pregnancy (Thatcher and Hansen, 1992). In such a context, embryo independent inhibition of uterine PGF2␣ synthesis should increase the survival of small or underdeveloped embryos (Binelli et al., 2001). Embryo independent inhibition of uterine PGF2␣ synthesis has been tried through administration of gonadotropin releasing hormone (Machado et al., 2008), human chorionic gonadotropin (Stevenson et al., 2007), progesterone (Mann et al., 2006), bovine somatotropin (Santos et al., 2004), recombinant bovine interferon-tau (Thatcher et al., 1997), flunixin meglumine (Guzeloglu et al., 2007), etc. Previous experiments both in vitro (Mattos et al., 2003) and in vivo (Oldick et al., 1997) have indicated the potential use of polyunsaturated fatty acids (PUFA) in decreasing PGF2␣ production from the uterus thereby reducing embryonic losses (Ambrose et al., 2005). Fish meal (FM) being used in dairy cow rations as a source of Rumen Undegradable Proteins (RUP), also contains a relatively high concentration of two polyunsaturated fatty acids of the n-3 family, eicosapentaenoic acid (EPA, C20;5) and docosahexaenoic acid (DHA, C22;6). FM supplementation, thus can improve pregnancy rates by inhibiting PGF2␣ secretion. Oxytocin stimulates secretion of PGF2␣ from uterine tissues when administered in vivo (Lafrance and Goff, 1985). Accordingly, the objective of the present study was to assess the effect of FM supplementation on secretion of PGF2␣ in dairy buffaloes. 2. Materials and methods 2.1. Experimental animals Twelve, apparently healthy, Murrah buffaloes in their second to fifth parity were selected for the study. They were kept under a loose housing system, comprising of concrete sheds and open space to walk around. The animals were fed ad libitum with seasonal green fodder and wheat straw. The buffaloes were provided with fresh drinking water thrice daily. Seven buffaloes were kept on routine feeding schedule (Control buffaloes, Group I) while five buffaloes were supplemented with 250 g FM/buffalo/day (Treatment buffaloes, Group II) for 55 days in addition to their routine feeding. The fish meal was fed @ 100 g/buffalo/day for the first week (acclimatisation period). On dry matter basis, FM contained 25.40% crude protein, 6.29% crude fibre, 5.22% ether extract and 64.13% ash. The total lipid content (5.86%) and free fatty acids (1.06%) of FM was determined as per the methods described by Folch et al. (1957) and Lowry and Tinsley (1976), respectively. 2.2. Estrus synchronization Both control and supplemented buffaloes (after 30 days of FM feeding) were estrous synchronized using Ovsynch protocol, similar to that developed for cattle (Pursley et al.,

1995) and previously applied in buffaloes (Neglia et al., 2003). Briefly, it involved the i.m. administration of a GnRH agonist (buserelin acetate, 20 ␮g; Receptal® , Intervet, Pune, India), irrespective of the stage of the estrous cycle followed by an i.m. injection of PGF2␣ (Cloprostenol Sodium; 500 ␮g VetmateTM , Vetcare, Bangalore, India) 7 days later, and second GnRH injection (20 ␮g) about 48 h after PGF2␣ (Fig. 1). 2.3. Corpus luteum size Ovaries were scanned trans-rectally using a real time B-mode diagnostic ultrasound scanner (MCV Concept, Scotland) equipped with a 7.5 MHz linear-array transducer. The diameter of corpus luteum was determined on day 12 of synchronized estrous cycle (day 0 = day of second GnRH; Fig. 1). Ultrasonographic assessments were made by the same person throughout the experiment. Optical scan images were frozen and the size of CL was determined by measurement of the diameter of the CL at their widest poles. 2.4. Blood sampling Blood samples were collected at 15 day interval for measurement of plasma glucose and total protein, during the study period. Plasma progesterone (P4 ) from blood samples collected on days 0, 5 and 12 of the synchronized estrous cycle (Fig. 1). Samples (10 ml) were collected by jugular venipuncture into heparinised polystyrene tubes (1:1000), immediately stored in ice and centrifuged at 1500 × g at 4 ◦ C for 15 min. Plasma was harvested and stored at −20 ◦ C until analysed. For plasma glucose estimation blood samples (5 ml) were taken separately in sodium fluoride (2.5 g/ml). 2.4.1. Oxytocin challenge Release of PGF2␣ was stimulated by intravenous injection of oxytocin (100 IU; Pitocin® , Pfizer, Mumbai, India) on day 15 of synchronized cycle. Blood samples (5 ml) were collected every 15 min for 5 h starting 1 h before to 4 h after oxytocin injection (−60, −45, −30, −15, 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240 min) by fixing a polyethylene catheter 1.0 mm diameter in jugular vein of each animal. Blood samples were centrifuged, plasma was harvested and stored at −20 ◦ C till further analysis. 2.5. Blood hormone and metabolite analysis The plasma concentrations of progesterone were estimated using modified liquid phase radioimmunoassay (Kamboj and Prakash, 1993). Sensitivity of the assay was 0.1 ng/ml; intra-assay and inter-assay variation coefficients were 6.2% and 9.5%, respectively. The concentrations of PGFM were estimated using a commercially available enzyme immunoassay kit (Cayman Chemical Company, Ann arbour, MI, USA). The detection limit of the assay was 12.8 pg/ml. Intra- and inter-assay variation coefficients for PGFM were 23.3% and 15.2%,

A.A. Malik et al. / Animal Reproduction Science 126 (2011) 45–49

47

Feeding FM @ 250 g/day

Days (-47

-40

-25

-10 -9

BS

A

-2

0

GnRH1 PG GnRH2

5

12

US

15)

OT

Fig. 1. Sequence of injections and collection of samples. A: acclimatisation; GnRH: (Receptal® ); PG: prostaglandin (VetmateTM ); US: ovarian ultrasound scan; OT: oxytocin challenge test. Dots (•); indicate days of blood sampling.

respectively. Plasma glucose and total protein were analysed using commercial biochemical assay kits on an Microlab-300 autoanalyzer (Merck specialities Private Ltd., Mumbai, India). 2.6. Statistical analysis The data obtained was analysed using Student’s t-test and Duncan’s multiple range test (Snedecor and Cochran, 1994) using SPSS-16 software. Differences were considered significant at P < 0.05. Fig. 2. Mean hourly plasma PGFM concentration (mean ± S.E.M.; pg/ml) for FM supplemented and control buffaloes following oxytocin challenge on day 15 of synchronized estrous cycle.

3. Results 3.1. Plasma 13,14-dihydro-15-keto-prostaglandin F2˛ The mean PGFM concentrations are presented in Table 1 and Fig. 2. Due to individual variation in PGF2␣ release after oxytocin challenge, the mean hourly concentration of PGFM was considered for its comparisons between FM supplemented and control groups. The plasma concentrations of PGFM were significantly lower (P < 0.05) in Group II buffaloes at −1 h of oxytocin challenge as compared those in Group I buffaloes (176.7 ± 31.5 pg/ml versus 266.4 ± 22.9 pg/ml). Within 1 h of oxytocin administration, PGFM concentration were more than mean basal concentration of PGFM indicating response of the animals to oxytocin injection. The basal concentration was the mean PGFM concentration from −1 h to 0 min relative to oxytocin injection (Wuenschel, 2006). A significant difference existed in mean concentrations of PGFM (p < 0.05) at 1 h (197.4 ± 41.7 pg/ml versus 326.3 ± 33.5 pg/ml) and 2 h Table 1 Hourly plasma PGFM concentration (Mean ± S.E.M.) and percent release in FM supplemented and control buffaloes following oxytocin challenge on day 15 of synchronized estrous cycle. Time

176.7 197.4 178.3 175.9 186.7

± ± ± ± ±

31.5a 41.7a (11.73) 30.7a (0.91) 38.2 (−0.4) 47.3 (5.6)

Control (n = 7) 266.4 326.3 289.5 288.3 275.6

3.2. CL size, plasma progesterone and metabolites The diameter of corpus luteum on day 12 (mean ± S.E.M.; 13.7 ± 1.0 mm versus 15.8 ± 1.4 mm) was not statistically different between FM supplemented and unsupplemented buffaloes. Plasma progesterone concentrations were similar in buffaloes of both the groups on days of sampling (Table 2). The mean values of Plasma glucose and total proteins on day 0, 15 and 30 of the study period remained within the normal physiological range are presented (Table 3). Table 2 Plasma progesterone concentration (Mean ± S.E.M.) in FM supplemented buffaloes and control.

PGFM concentration (pg/ml) following oxytocin challenge Supplemented (n = 5)

−1 h 1h 2h 3h 4h

(178.3 ± 30.7 pg/ml versus 289.5 ± 31.3 pg/ml) post oxytocin challenge in FM supplemented and control group buffaloes, albeit PGFM concentrations were numerically lower in the former at all occasions. The percent release of PGFM was double in control animals compared to supplemented ones after oxytocin challenge (22.47% versus 11.73%). The basal and peak concentrations of PGFM did not differ significantly (P < 0.05) within the groups.

± ± ± ± ±

22.9b 33.5b (22.47) 31.3b (8.66) 39.7 (8.19) 41.8 (3.43)

Means within row with different superscripts differ (P < 0.05). Values in parenthesis indicate percentage change in release of PGFM.

Day of cycle

0 5 12

Progesterone Concentration (ng/ml) Treatment (n = 5)

Control (n = 7)

0.23 ± 0.058 0.49 ± 0.12a 1.10 ± 0.22b

0.38 ± 0.07a 0.50 ± 0.1a 1.05 ± 0.27b

a

In columns mean values having different superscript differ significantly (P < 0.05).

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A.A. Malik et al. / Animal Reproduction Science 126 (2011) 45–49

Table 3 Plasma glucose and total protein concentration (Mean ± S.E.M.) in FM supplemented and control buffaloes. Metabolic parameter

Group

Days of FM supplementation 0

15th

30th

Glucose (mg %)

Supplemented (n = 5) Control (n = 7)

60.1 ± 1.6 60.3 ± 2.8

64.9 ± 2.1 59.7 ± 2.3

63.5 ± 2.4 62.5 ± 1.8

Total protein (g %)

Supplemented (n = 5) Control (n = 7)

7.6 ± 0.2 7.8 ± 0.2

7.8 ± 0.4 7.9 ± 0.4

7.6 ± 0.5 7.6 ± 0.2

Differences in mean values with in and between groups are non significant (P < 0.05).

4. Discussion The present study is among the first to assess the antiluteolytic effect of fish meal supplementation in dairy buffaloes. PGF2␣ is rapidly metabolised through the pulmonary system in most species (Weems et al., 2006), hence plasma concentrations of its more stable metabolite (PGFM) were measured. Average basal PGFM concentration on day 15 of synchronized cycle observed in Group I buffaloes were similar to values as reported earlier (Batra and Pandey, 1983) and much higher as compared to values found during mid luteal phase (Mishra et al., 2003). The low concentrations of PGFM at −1 h in the Group II buffaloes indicate attenuation in release of PGF2␣ by FM supplementation over 55 days. Mattos et al. (2004) reported significant reductions in PGFM concentrations in early postpartum dairy cows fed fish oil. A significant attenuation (P < 0.05) in the release of PGFM after OT-challenge was observed in FM supplemented animals (Fig. 2). Mattos et al. (2002) reported significant reductions in PGFM concentrations following an oxytocin challenge on day 15 of a synchronised oestrous cycle in dairy cows fed fish meal. Contrary to our findings, others (Wamsley et al., 2005; Heravi Moussavi et al., 2007) found no effect or even increased PGFM secretion (Petit et al., 2002; Childs et al., 2008) following oxytocin administration on day 15 in lactating cattle supplemented with FM. This inconsistent pattern may be related to amount of n-6 fatty acids reaching the target tissue, i.e. endometrium to cause the net inhibition of PGF2␣ by n-3 fatty acids (Heravi Moussavi et al., 2007) and slight changes in cycle length between animals which may confound the treatment differences (Wathes et al., 2007). Different mechanisms have been suggested in the decrease of prostaglandin production by FM supplementation including decreasing the availability of precursor arachidonic acid (Christiansen et al., 1991), increasing the concentration of fatty acid that compete with arachidonic acid for processing by prostaglandin-H synthase enzyme (Chen and Nilsson, 1993) and inhibition of prostaglandinH synthase enzyme synthesis and activity (Achard et al., 1997). Also prostaglandin-H synthase converts EPA into prostanoides of the series 3 in a less efficient manner than it converts arachidonic acid into prostanoides of the 2 series. A lower efficiency of catalysis may result in reduced total prostanoid synthesis. No effect of FM supplementation was observed on plasma progesterone concentration on days 0, 5, and 12 of the synchronized cycle. Different workers have reported an increase (Burke et al., 1997), decrease (Robinson et al., 2002) or no effect (Mattos et al., 2002; Wamsley et al., 2005)

of FM supplementation on plasma progesterone concentrations of dairy animals. Similarly FM supplementation did not affect the size of mid-cycle CL. Lipid supplementation had no effect (Petit et al., 2001) or increased the size of the corpus luteum as might be expected from reports on n-3 PUFA intake (Petit et al., 2002; Childs et al., 2008). This discrepancy in results may be due to the differences in the dietary fatty acid profile as well as energy status of the animals. The importance of glucose as an energy source for the bovine ovary and the post-blastulation embryo has been well documented (Boland et al., 2001). In the present study, there was no effect of FM supplementation on systemic glucose concentrations. Increased (Heravi Moussavi et al., 2007), decreased (Mattos et al., 2004), consistent (Petit et al., 2002) systemic glucose concentrations in dairy cows fed FM supplements have been reported in literature. Grummer and Carroll (1991) reviewed the literature and concluded that fat supplementation does not routinely increase blood glucose and that stable systemic glucose concentrations during fat supplementation may indicate a reduction in hepatic gluconeogenesis. Plasma protein concentration remained similar in buffaloes of both the groups. Previous studies are lacking to substantiate the present findings of plasma total protein levels with respect to the FM supplementation. 5. Conclusions It is concluded that, fish meal supplementation decreased the uterine secretion of PGF2␣ in response to oxytocin challenge on day 15 of estrous cycle without affecting CL size and plasma progesterone concentrations in dairy buffaloes. Acknowledgements This work was funded by National fund for Basic and Strategic Research in Agriculture (NFBSRA), ICAR, New Delhi under the project ‘Antiluteolytic strategies, a novel approach to enhance fertility in buffalo’. References Achard, D., Gilbert, M., Benistant, C., Slama, S.B., DeWitt, D.L., Smith, W.L., Lagarde, M., 1997. Eicosapentaenoic and Docosahexaenoic acids reduce the PGH synthase expression in bovine aortic endothelial cells. Biochem. Biophys. Res. Commun. 241, 513–518. Ambrose, D.J., Kastelic, J.P., Corbett, R., Pitney, P.A., Petit, H.V., Small, J.A., Zalkovic, P., 2005. Lower pregnancy losses in lactating dairy cows fed a diet enriched in linolenic acid. J. Dairy Sci. 89, 3366–3374.

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