Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality

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Theriogenology 76 (2011) 419 – 426 www.theriojournal.com

Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality V. Carcangiu*, M.C. Mura, M. Pazzola, G.M. Vacca, M. Paludo, B. Marchi, C. Daga, S. Bua, S. Luridiana Dipartimento di Biologia Animale, Università degli Studi di Sassari, Via Vienna 2, 07100, Sassari Received 25 October 2010; received in revised form 4 February 2011; accepted 15 February 2011

Abstract The aim of this study was to examine the polymorphism in MTNR1A gene and its relation to reproductive seasonality in Mediterranean Italian buffaloes reared in Sardinia. The mating period and calving of 100 multiparous buffalo-cows were recorded for three years (2005–2008). Genomic DNA was subjected to PCR for the amplification of the exon II, then 40 amplicons were sequenced. The obtained sequence was deposited in GeneBank database (accession number GU817415). PCR products were checked for the presence of HpaI restriction sites and assigned to genotypes “C/C”, “C/T” or “T/T”. Allelic frequency of C and T alleles was 0.44 and 0.56 and genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T. In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January and their calving mainly occurred from August to September. On the other hand animals with T/T genotype showed mating mostly in the semester between February and July and calving occurred largely from March to May in all the three years. Heterozygous, in all the three years, showed about the same number of animals mated within each six-month period. The results of the present study provide for the first time a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from buffaloes. Moreover our data showed an association between Single Nucleotide Polymorphism and seasonal reproductive activity in these animals. © 2011 Elsevier Inc. All rights reserved. Keywords: Buffaloes; MTNR1A gene; Reproductive seasonality

1. Introduction Buffaloes living under Mediterranean latitudes can be considered to have a tendency to be seasonal breeding animals and their reproductive efficiency is usually negatively affected by increasing day-length which consequently influences productions [1,2]. Photoperiod, through the melatonin secretion, is the main en-

* Corresponding author. Tel.: ⫹39079229436; fax ⫹39079229592. E-mail address: [email protected] (V. Carcangiu). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.02.018

vironmental factor affecting the regulation of reproductive seasonality [3,4]. Melatonin is produced by pineal gland at night in direct proportion to the period of darkness [5]. The pattern of melatonin secretion provides photoperiodic information to cells within the brain that possess the relevant receptors and control reproductive function [6]. Melatonin receptors are classified in MTNR1A and MTNR1B subtypes but only the first seems to be involved in the regulation of seasonal reproductive activity [7,8]. The MTNR1A receptor gene in sheep is on chromosome 26, in cattle on chro-

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mosome 27 and in buffalo on chromosome 1 which is a fusion of Bos Taurus chromosome 1 and 27 [9,10]. The melatonin effect is carried out at hypothalamic level, by regulating of GnRH secretion [11]. However the highest concentration of melatonin MTNR1A receptors has been evidenced in the Pars Tuberalis (PT), but this region seems to be particularly involved in the control of Prolactin secretion [12,13]. Inversely, in the Premammillary Hypothalamus (PMH), low density of melatonin binding sites was found (20 –100 times lower than in the PT), but functional studies have shown that melatonin micro-implants placed into PMH are able to stimulate the GnRH system [11,14]. In several sheep, goat and cattle breeds, polymorphic sites in MTNR1A receptor gene exon II were found [15]. One G to an A substitution in position 612 in sheep and a G to an A substitution in position 52 in goat of the sequence of MTNR1A receptor gene lead to a less seasonal reproductive activity [16,17]. Polymorphic site was found in cattle but no correlation is known with reproductive activity [15] whereas in buffalo it is still unknown if there are polymorphisms of the melatonin receptor gene and whether there are relationships with seasonality of reproduction. In Italy buffaloes are bred for the main purpose of producing and marketing milk and its derivatives of which mozzarella cheese is probably the best known worldwide [18]. Mediterranean Italian buffalo-cows show a decline in reproductive activity from mid-winter to spring in response to increasing daylength [19]. The seasonal decline in reproductive activity is manifested by a reduced incidence of estrous behaviour, a decrease in the proportion of females that undergo regular estrous cycles and a generally lower conception rate [19]. In the Mediterranean region it is necessary to plan the mating of buffaloes during the seasonal trough in reproduction so that calving coincides with the annual peak for buffalo milk demand [20]. Subsequently, a strategy has been developed in Italy in order to reverse the calving season in buffalocows and it has been termed the Out-of-BreedingSeason-Mating (OBSM) technique [21]. This technique is applied by removing bulls from the herd in October and reintroducing them between March and the end of September so that most calving occurs between the end of January and the beginning of August. Over the years the implementation of the OBSM technique has selected animals less sensitive to daylight variation and explained the differences found with respect to the nocturnal and seasonal variation of melatonin [22]. Therefore it could be of great interest to identify a suitable method to recognize buffaloes less sensitive to

photoperiod. Thus, the aim of the present research was firstly to study the polymorphism in MTNR1A gene and then to emphasize its association with seasonal reproduction of the Mediterranean Italian buffaloes reared in Sardinia. 2. Materials and methods 2.1. Experimental design This study was undertaken in a homogeneous herd of about 300 Mediterranean Italian buffaloes, located in the South of Sardinia (39° 36’ N). All buffaloes were under natural photoperiod and housed in large open yards with sheltered areas. The daily feed allocation consisted of 5 kg ryegrass Italian hay, 18 kg corn silage (30% dry matter), 2 kg soybean meal (44% crude protein), 4 kg grain mix (22% crude protein), 2 kg corn meal and 0.1 kg hydrolyzed fats. The study was conducted using 100 multiparous buffalo-cows which were 6.24 ⫾ 1.20 years old (range: 4 – 8). Considering the age influence on reproductive activity, the primiparous and the old cows were excluded from the study. Reproductive activity of the last three years (2005–2008) was recorded for each animal. All the buffaloes included in the study were in good general health and without reproductive disorders. Bulls (1:25 male/ female ratio) were kept always within the herd. Earmark numbers of the estrous and mated females were recorded by trained technicians. Estrous detection was performed by observing estrous-behaviour (marked by bellowing, homosexual mounting, being sniffed, mounted, or serviced by the male). The pregnancy checking was performed by palpation per rectum and/or ultrasound between days 40 and 60 post-mating using an Esaote Piemedical Tringa linear equipment (Esaote Europe B.V., Maastricht, The Netherlands) provided with a 5.0 –7.5 MHz multiple frequency linear probe. 2.2. Genomic DNA preparation 10 mL of blood was collected from the caudal vein of each buffalos using a tube with EDTA as an anticoagulant (Believer Industrial Estate, Plymouth, UK). Genomic DNA was extracted from whole blood, using a commercial kit (NucleoSpin Blood QuickPure, Macherey-Nagel, Duren, Germany) and then kept at ⫺20 °C until use. 2.3. Primer sequences 100 –150 ng of genomic DNA were used for PCR reaction using primers by Messer et al. [15], sense

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Fig. 1. Partial sequence of exon II MTNR1A gene and deduced amino acid sequence in Mediterranean Italian buffalo and cattle. Black arrow: HpaI cleavage site; Black bars: nucleotide changes; Gray bars: amino acid changes. The buffalo sequence has been deposited in GenBank under accession number GU817415.

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primer 5=–TGT GTT TGT GGT GAG CCT GG–3= and antisense primer 5=–ATG GAG AGG GTT TGC GTT TA–3=. Primers were synthesized by Sigma Genosys Ltd (Pampisford, Cambs, UK). 2.4. PCR methods Reaction of PCR was carried out in 50 ␮L volume, containing 10x PCR Buffer (50 mmol L⫺1 KCl, 10 mmol L⫺1 Tris-HCl (pH 8.0), 0.1% (wt/vol) Triton X-100) 5␮l, 1.5 mmol L⫺1 MgCl2 3␮L, 0.2 mmol L⫺1 each dNTP 8␮L, 10 pmol L⫺1 each primer 1␮L, 100 – 150 ng bufaline genomic DNA and 5U Taq DNA polymerase (HotMaster Taq DNA Polymerase, Eppendorf AG, Hamburg Germany). PCR conditions were as follows: denaturation at 94 °C for 5 min followed by 35 cycles shared in denaturation 94 °C for 1 min, annealing 62 °C for 1 min, extension 72 °C for 1 min and final extension 72 °C for 10 mins, on Mastercycler® Gradient (Eppendorf AG, Hamburg, Germany). PCR products were separated by electrophoresis on 2% (wt/vol) agarose gel (GellyPhor, Euroclone, UK), in parallel with 100 bp DNA marker (Invitrogen, Carlsbad, CA, USA).

DNA fragments in recombinant plasmids were then sequenced from both directions using Applied Biosystems 3730 DNA Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA), with Dye Terminator 3.1 chemistry. In the second procedure, 30 ␮L from another 20 samples of PCR products were purified using a magnetic support (MagnaRack®, Invitrogen, Carlsbad, CA, USA) and a purification kit, ChargeSwitch® PCR Clean-Up Kit (Invitrogen, Carlsbad, CA, USA). The resulting samples were again sequenced from both directions. 2.6. Sequence alignments and restriction enzyme digestion Sequences were aligned with bovine MTNR1A receptor gene sequence (GenBank accession numberU73327) to evidence nucleotide differences. Then buffalo MTNR1A receptor gene sequence was deposited to GenBank. All the PCR products were digested using 2U of HpaI enzyme (New England Biolabs, Beverly, MA, USA). The digestion reaction was carried out in 30␮L volume, containing PCR product 20 ␮L, BSA

2.5. Cloning and sequencing Two cDNA preparation procedures were performed before sequencing. In the first procedure the resulting fragment from PCR of 20 samples were ligated into the pCR®-4 TOPO vector (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions at 16 °C overnight. The ligation reaction was carried out in 12 ␮L volume, containing PCR product 9 ␮L, pCR®-4 TOPO vector 1␮L (10 ng/␮L in 50% (v:v) glycerol, 50 mmol L⫺1 Tris HCl, pH 7.4, 1 mmol L⫺1 EDTA, 2 mmol L⫺1 DTT, 01% (wt/vol) Triton X-100, 100␮g/mL BSA, 30 ␮mol L⫺1 phenol red), Salt Solution (1.2 mmol L⫺1 NaCl, 0.06 mmol l⫺1 MgCl2) 2␮L. Each fragment was transformed into Escherichia Coli DH5␣ competent cells. From recombinant bacterial colonies, plasmid was extracted using Perfectprep® Plasmid Mini Procedure Kit (Eppendorf AG, Hamburg, Germany). Then, to confirm cloning of recombinant vector, a control digestion was performed using EcoRI restriction enzyme (England Biolabs, Beverly, MA, USA). The digestion reaction was carried out in 20 ␮L volume, containing EcoRI enzyme 10U, recombinant vector 8␮L, NEBuffer 1x (100 mmol L⫺1 Tris HCl, 50 mmol L⫺1 NaCl, 10 mmol L⫺1 MgCl2, 0.025 Triton X-100, pH 7.5) 2␮L, at 37 °C for 2 h, followed by a process of enzyme deactivation performed at 65 °C for 20 mins.

Fig. 2. Electrophoresis of digestion with HpaI on a 4% agarose gel in the Mediterranean Italian buffalo. Lane 1 and 8: 100 bp DNA marker. Lane 2 and 3: cleavage site present (genotype C/C: bands of 745 bp and 79 bp); Lane 4 and 5: cleavage site absent (genotype T/T: a band of 824 bp); Lane 6 and 7: cleavage site present in only one parental chromosome (genotype C/T: simultaneous presence of the 824 bp, 745 bp and 79 bp bands).

V. Carcangiu et al. / Theriogenology 76 (2011) 419 – 426 Table 1 Allele and genotype frequency in the Mediterranean Italian buffalo (n ⫽ 100) allele C 0.44

genotype T

C/C

C/T

T/T

0.56

26%

40%

34%

(100 ␮g/mL) 0.3 ␮L and Buffer 1x (10 mmol L⫺1 Tris-HCl, 50 mmol L⫺1 NaCl, 10 mmol L⫺1 MgCl2, 1 mmol L⫺1 Dithiothreitol, pH 7.9) 3␮L, at 37 °C for 2 h, followed by a deactivation process led at 65 °C for 20 mins. Resulting fragments were separated by electrophoresis on 4% (wt/vol) agarose gel (GellyPhor, Euroclone, UK), in parallel with a 50 bp DNA marker (Invitrogen, Carlsbad, CA, USA). Genotyping was performed on all the 100 samples. 2.7. Statistical analysis Genotypic frequencies, allelic frequencies, and Hardy–Weinberg equilibriums were calculated using Genepop. Data was analysed using the Fisher’s exact test and ␹2_test in order to evaluate the link of genotype with the number of mated and calved animals according to season. 3. Results PCR product resulted in a 824 bp fragment corresponding to the main part of the exon II of the MTNR1A melatonin receptor gene (GeneBank accession number GU817415). Buffaloes MTNR1A gene sequencing showed an over 97% nucleotide identity with bovine sequence, as only 23 nucleotide differences (of which 9 leading to amino acid changes) were registered between the two species (Fig. 1). The 40 buffaloes sequence alignment made it possible to detect a polymorphism in position 82 as characterized by the presence of a C or a T. Several restriction enzymes were tested and only HpaI was able to identify the above mentioned mutation. The presence of the cleav-

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age site was caused by the presence of C and after electrophoresis it produced one band of 79 and one of 745 bp (Fig. 2). The absence of this cleavage site was caused by the presence of T which leaves the uncut 824 bp fragment (Fig. 2). With the presence of 824bp, 745 and 79 bp bands, the genotype is C/T (Fig. 2). Frequency of C and T alleles was respectively 0.44 and 0.56 in the analyzed population which is in Hardy Weinberg equilibrium. The genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T (Table 1). In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January (Table 2) and their mating peak has been recorded between October and November. The pregnancy status was confirmed by palpation per rectum and/or ultrasound between days 40 and 60 post-mating. Consequently calving in C/C animals mainly occurred from August to September in all the three years (Fig. 3). However, animals with T/T genotype mated mostly in the semester between February and July (Table 2). Their mating peak has been recorded between May and July (Table 2) and calving occurred largely from March to May in all the three years (Fig. 3). While the heterozygous, in the corresponding three years, showed that about the same number of animals mated within each six-month period displayed in Table 2. All the animals mated in the same period during all of the three years with the only exception of four cows of C/C and one of T/T genotype. The exceptions of C/C genotype buffalos were No. 235, No. 481, No. 1052 and No. 1187 in the year 2006 and No. 235 in the year 2007 which mated between February and July instead of between August and January. An analogous change was found in buffalo No. 1216 of T/T genotype in the year 2007, which mated between August and January. In the remainder of the animals a small variation of advance or delay of a few weeks was recorded in mating dates, but always within the same considered period.

Table 2 Number of Mediterranean Italian buffaloes mated in the three years according to HpaI genotype HpaI genotype Mating period Animals mated in 2005/06 Animals mated in 2006/07 Animals mated in 2007/08

C/C

C/T

T/T

Aug-Jan

Feb-July

Aug-Jan

Feb-July

Aug-Jan

Feb-July

20 16 17

6 10 9

23 22 23

17 18 17

10 10 11

24 24 23

For 2005/06 ␹2 test ⫽ P ⬍ 0.001; for 2006/07 and 2007/08 ␹2 test ⫽ P ⬍ 0.05.

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4. Discussion

Fig. 3. Number of calving recorded monthly according to C/C (●) and T/T () genotype in each observed year.

The partial nucleotide sequence encoding the MTNR1A receptor in buffalo (GenBank accession numbers GU817415) showed the same number of nucleotides found in cattle, sheep and goats [15,17]. As expected, the highest similarity (over 97 %) was found between the bovine and bufaline sequence, since only 23 nucleotide changes have been registered. This is justified by the phylogenetic proximity between these two species belong to the Bovidae family. The karyotype between the buffalo and domestic cattle are in fact very similar, although there are 60 chromosomes in cattle and 50 in Mediterranean Italian buffalo and this difference is due to the fusion of different chromosomes [10]. Our data about multiple buffaloes sequence alignments highlights the existence of a polymorphic site in position 82, and also, in sheep, goat and cattle polymorphic sites were found in different positions from these in buffaloes [15,16]. This polymorphism has been related to reproductive seasonality in some ovine and caprine breeds only [23,24]. In our data the animals homozygous for allele T showed reproductive activity during increasing photoperiod while those with genotype C/C reproduced largely during decreasing photoperiod. Generally, at Mediterranean latitudes, buffaloes manifested their sexual activity mainly when daylight hours decrease and show a poor reproductive efficiency from mid-winter to spring, when daylight hours increase [19]. This aspect is of particular importance in some countries, like Italy, where market demand for buffalo milk coincides with the months of the year when calving is less frequent. So it is important to plan calving between February and June with the main aim of producing milk for processing into mozzarella cheese in spring and early summer. Buffaloes which calve between August and January produce 60 –90 % of their milk during the period of lower market demand [25]. On the basis of our data we can speculate that the individual sensitivity to photoperiod could be due to the MTNR1A genotype. In fact buffaloes carrying C/C genotype showed the reproductive activity principally during decreasing day-length whereas those with T/T genotype showed mating period largely during increasing day-length. Consequently, our results can provide valuable support for improve the reproductive efficiency during non breeding season in buffaloes. The genetic identification of the animals is the first step for the improvement of farm reproductive management. In fact the OBSM technique could have a better success if the buffalo’s genotype is known. Undoubtedly animals carrying T/T genotype could be allocated to reproduc-

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tion during long photoperiod instead the C/C subjects during natural mating season. Therefore the identification from birth of the MTNR1A genotype in buffaloes drives to an early selection, avoiding the wait for the adult reproductive performances. Thus the polymorphism studied here should be considered possible genetic markers suitable for increasing farms profitability. However, it is difficult to explain the mechanism underlying the association found between genotype and reproductive seasonality in the Mediterranean buffalo. The most plausible hypothesis is that this polymorphism is associated with other mutations in other parts of the MTNR1A gene determining an alteration in the signalling pathway as recently found in sheep [26]. In fact in this specie the silent mutation G612A is associated with the non conservative G706A and changes a Valine in an Isoleucine (Val220Ile) altering the cAMP signal transduction pathway. This suggests a potential modification in the interpretation of the melatonin signal and could participate to the phenotypic differences of reproduction among the different genotypes [26]. Therefore, the same hypothesis could be formulated for buffaloes and would also explain the different reproductive seasonality found in the present research. In conclusion, the results of the present study provide, for the first time, a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from Mediterranean Italian buffaloes. Moreover, our study evidenced that, in these animals the two allelic forms have direct effect on the seasonal reproductive activity. To interpret the significance of this finding it will be necessary in the future to examine, in both genotypes, other parameters of functional importance such as those involved in the signalling pathway. The study of the relationship among the several discovered mutations or the finding of new mutations in other region of the MTNR1A gene, can provide useful information to explain the mechanism influencing the seasonal reproduction. The discovered association between the T/T genotype and reproductive activity during long photoperiods indicates that this polymorphism can be considered a genetic marker to identify buffaloes able to reproduce out of breeding season.

Acknowledgments The authors are grateful to the owner of the SOGESTIM s.r.l. farm for their helpfulness. This work was supported by a grant from the Foundation of Banco di Sardegna and Regione Autonoma della Sardegna (L.R. 7/2007).

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