| Abstract | U radu je istraživan utjecaj polimorfizama na lokusu 606 i lokusu 612 MTNR1A gena na
sezonsku poliestričnost ovaca. Ciljevi predmetnog istraživanja bili su: 1) utvrditi
frekvencije genotipova i alelnih varijanti MTNR1A gena na pozicijama 606 i 612 referentne
sekvence U14109, 2) utvrditi prisutnost polimorfizama na istraživanoj sekvenci drugog
egzona MTNR1A gena, 3) utvrditi utjecaj polimorfizama MTNR1A gena na sezonsku
poliestričnost, dob ovaca pri prvom janjenju i dužinu međujanjidbenog razdoblja.
Prikupljanje biološkog materijala (krv, tkivo, dlaka) i reprodukcijskih pokazatelja ovaca
provedeno je tijekom 2015. i 2016. godine na području Republike Hrvatske i Republike
Slovenije ovisno o mjestu uzgoja pojedine pasmine. U Hrvatskoj su prikupljeni uzorci od
hrvatskih autohtonih pasmina ovaca: cigaje, dalmatinske pramenke, dubrovačke ovce,
istarske ovce, ličke pramenke i paške ovce, zatim uvezenih pasmina ovaca:
istočnofrizijske ovce, romanovske ovce i Suffolka, dok su u lovištu nakon usmrćivanja
(odstrela) prikupljeni uzorci muflona. U Sloveniji su prikupljeni uzorci od slovenske
autohtone pasmine: bovške ovce. Izuzev plemenitih pasmina ovaca koje odlikuje
produženo razdoblje pripusne sezone, preostale pasmine odlikuje sezonska pojava
spolnog ciklusa te slove kao sezonski poliestrične. Ukupno je prikupljeno 439 uzoraka iz
kojih je napravljena izolacija genomske DNK. Nakon izolacije genomske DNK sa
specifičnim začetnim oligonukleotidima amplificiran je glavni dio drugog egzona MTNR1A
gena dužine 824 bazna para koji je podvrgnut enzimatskoj razgradnji. Za detekciju
polimorfizama na lokusima 606 i 612 korištena je RsaI odnosno MnlI restrikcijska
endonukleaza. Genotipizacija uzoraka bila je sljedeća: CC, CT i TT na lokusu 606 prema
RsaI restrikcijskoj endonukleazi te GG, GA i AA na lokusu 612 prema MnlI restrikcijskoj
endonukleazi. U pet uzoraka svakog genotipa utvrđenog MnlI restrikcijskom
endonukleazom određen je slijed nukleotida na ciljnom odsječku istraživane DNK i
potvrđena prisutnost polimorfizama na istraživanim lokusima (606 i 612) te detektirano 6
dodatnih mutacija. Polimorfizam na lokusu 606 se očitovao prisutnošću citozina (alel C)
umjesto timina (alel T), dok se polimorfizam na lokusu 612 očitovao prisutnošću gvanina
(alel G) umjesto adenina (alel A). Preostalih šest mutacija bile su: G453T, G706A, G783A,
G801A, C891T i C893A. Na lokusu 606 MTNR1A gena u muflona, romanovske i Suffolk
pasmine utvrđene su visoke frekvencije alela C dok su u sezonski poliestričnih pasmina
utvrđene veće frekvencije alela T. Na lokusu 612 u gotovo svih istraživanih pasmina
ovaca, utvrđena je visoka frekvencija alela G (od 0,50 do 0,95) izuzev muflona u kojeg je
utvrđena visoka frekvencija alela A (0,93). U istraživanih ovaca utvrđena su janjenja u
svim istraživanim sezonama, a bez obzira na genotip na lokusu 606 i 612 najveći broj
janjenja je utvrđen tijekom zime (61,89%). Po broju janjenja tijekom ljeta dominirali su
homozigoti (CC/606; GG/612) i heterozigoti (CT/606; GA612) za oba promatrana lokusa.
Rezultatima predmetnog istraživanja nije utvrđen statistički značajan utjecaj polimorfizama
na lokusu 606 i lokusu 612 MTNR1A gena na sezonsku poliestričnost ovaca, dob pri
prvom janjenju niti na prosječnu dužinu međujanjidbenog razdoblja. Prisutnost
polimorfizama na lokusu 612 povoljno je djelovala na pojavu janjenja izvan uobičajene
sezone (ljeto i/ili jesen) u cigaje, istarske ovce, ličke pramenke i romanovske ovce, zatim
na janjenja s manjom dobi u cigaje, dalmatinske pramenke, istočnofrizijske ovce, ličke
pramenke i romanovske ovce te na kraće međujanjidbeno razdoblje u cigaje, dubrovačke,
istarske, istočnofrizijske i romanovske ovce. |
| Abstract (english) | Seasonal reproduction is a common feature among various mammalian species of the
temperate latitudes. Ewes are well known for seasonal breeding activity which usually
appears at the end of summer or at the beginning of autumn and finish in late winter or at
the very beginning of spring. The seasonality of reproductive activity in sheep breeds in
temperate latitudes is controlled by the photoperiod. In sheep, short photoperiods
stimulate pineal gland to synthesize hormone melatonin, while long photoperiods inhibit
pineal gland in melanin synthesis. Therefore, melatonin inform animal on photoperiod
changes and has stimulating effect on hypothalamus which release Gonadotropinreleasing
hormone. Pituitary gland stimulated by hypothalamus release Luteinizing
hormone which affects the growth of oocytes. In sheep melatonin exerts its function
through specific MT1 G-protein receptor. Gene for this receptor (MTNR1A gene) is
located on chromosome 26 and has two polymorphic sites at position 606 and 612 which
are associated to aseasonal breeding activity. Considering that MTNR1A gene has been
proposed as candidate gene for out of season breeding in sheep, present study evaluates
the effects of polymorphisms at positions 606 and 612 on sheep seasonal reproduction.
The objectives of the study were: 1) determine the frequency of genotypes and allele
variants of MTNR1A gene at positions 606 and 612 (reference sequence U14109), 2)
determine the presence of polymorphisms on the studied sequences on the second exon
of MTNR1A gene, 3) determine the effect of MTNR1A gene polymorphisms on seasonal
reproduction, age at first lambing and interval between lambing. Depending on breeding
place the biological material (blood, tissue, hair) and sheep reproductive parameters were
collected during 2015 and 2016 year in the area of Republic of Croatia and Republic of
Slovenia. Samples of Croatian indigenous sheep breeds (Tsigai sheep, Dalmatian
Pramenka, Dubrovnik sheep, Istrian sheep, Lika Pramenka sheep and Pag Island sheep),
imported noble sheep breeds (East Friesian sheep, Romanov sheep and Suffolk), as well
as mouflon (Ovis musimon) samples (from hunting grounds) were collected in Croatia. In
Slovenia were collected samples of Slovenian indigenous breed: Bovec sheep. Except for
noble sheep breeds which are characterized by a prolonged period of mating season, the
rest of breeds express seasonal sexual activity and they are considered as seasonal
polyestric sheep breeds. From 439 collected samples genomic DNA was extracted using
GenElute® Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). After DNA extraction,
the main part of second exon MTNR1A gene was amplified using specific primers
(forward: 5'-TGTGTTTGTGGTGAGCCTGG-3'; reverse: 3'ATTTGCGTTTGGGAGAGGTA-5')
adopted from Messer et al (1997). Obtained sequence
length was 824 base pairs which was then subjected to enzymatic resolution with RsaI
and MnlI restriction endonucleases. Polymorphism at position 606 was detected using
RsaI, while polymorphism at position 612 was detected using MnlI restriction
endonucleases. Samples were genotyped as follows: CC, CT or TT on locus 606 and GG,
GA or AA on the locus 612 of MTNR1A gene. In five samples of each genotype
determined with MnlI restriction endonucleases nucleotide sequence were determined in
the target sequence of investigated DNA and confirmed the presence of polymorphisms in
the studied loci (606 and 612). Reproductive parameters were date of birth of studied
animal and dates of all her lambing records. Season of birth and seasons of lambings
were calculated on the basis of date of birth and dates of lambings. Date of birth and date
of first lambing were used to determine length of interval (in days) required for animal to
have first offspring. Dates of subsequent lambings were used to calculate the average
interval between lambing. For genotype effects estimation on first lambing on loci 606 and
612 the following linear model was used: Y1ijklm = µ + tri + srj + pk + Gl + eijklm. For
genotype effects estimation on interval between lambing on loci 606 and 612 the following
linear model was used: Y2ijklmn = β0 + β1xijklmn + tri + srj + pk + Gl + eijklmn. Considering the
length of restriction fragments observer on agarose gel under UV light all three genotypes
were detected at locus 606 and 612 of the MTNR1A gene in studied populations.
Genotype frequencies at locus 606 were: 0.26, 0.45 and 0.29 for CC, CT and TT,
respectively. Genotype frequencies at locus 612 were: 0.46, 0.39 and 0.15 for GG, GA
and AA, respectively. Allele frequencies were: 0.49 for C, 0.51 for T, 0.66 for G and 0.34
for A allele. Differences in determined genotype frequencies between mouflon and all
studied sheep breeds were statistically significant at both loci (P<0.001; P<0.05). Also
statistically significant differences in determined genotypes frequencies were found
between investigated sheep breeds. High frequency of the allele C on locus 606 was
determined in mouflon, Romanov and Suffolk breeds while seasonal polyestric sheep
breeds had higher frequencies of allele T. The locus 612 in almost all studied sheep
breeds revealed a high frequency of the allele G (0.50 to 0.95) except mouflon in which
we identified a high frequency of allele A (0.93). The most frequent combinations of
genotypes at position 606 and 612 were: CTGA (27.79%), TTGG (27.11% and CTGG
(16.86%). The examined populations were in Hardy-Weinberg equilibrium for both loci,
except for locus 612 in Bovec sheep (P<0.01) and on both loci (606 and 612) in Dubrovnik
sheep (P<0.01). The polymorphism at locus 606 was expressed by the presence of
cytosine (C allele) instead of thymine (T allele), while polymorphisms at loci 612 was
expressed by the presence of guanine (G allele) instead of adenine (A allele). Except
those two mutations another six mutation were detected at the investigated DNA
sequence. Those six mutations were: G453T, G706A, G783, G801, C891T and C893A.
The mutation in position G706A and C893A led to an amino acid change in position 220
and 282, while all other mutations were silent. Sheep lambing were recorded in all four
seasons, and regardless of genotype the highest number of lambing was recorded in
winter (61.89%). Although out of season (summer and fall) was recorded only 11% of
lambing, ewes with homozygous (CC/606; GG/612) and heterozygous (CT/606; GA/612)
genotype dominated. In spring lambing dominated genotype combination CTGA (32.37%),
in summer genotypes CCGG, CTGG CTGA with 25% each, in fall genotypes TTGG and
CTGA and in winter genotypes CTGA and TTGG. Age of first lambing regarding genotype
at position 606 was very uniform from 513.8 days in genotype CC to 519 days in genotype
TT. On the other side at position 612 animals with genotype AA needed 434.9 days to first
lambing, animals with GG genotype 525.1 days and GA genotype 521.6 days. Differences
between genotypes on both loci were not statistically significant. Combination CTAA had
the most favourable effect on lower age at first lambing. Individuals with mentioned
genotype lambed with average age of 375.5 days while individuals with CCGG genotype
were the oldest at first lambing with 587.9 days. Average interval between lambing in
observed genotypes were as follow: 360.4, 358.5 and 361.3 days in CC, CT and TT
genotype and 359.3, 360.7 and 356.6 days in GG, GA and AA genotype, respectively.
Differences between genotypes on both loci were not statistically significant. Combination
of genotypes TTAA had the smallest interval between lambing of 352.3 days while
genotype combination TTGA had the biggest lambing interval (394.1 days). In the end we
can conclude that polymorphisms at loci 606 and 612 of MTNR1A gene were observed in
studied populations. Despite the absence of statistically significant effect of genotypes at
loci 606 and 612 on studied parameters some positive effect of MTNR1A gene
polymorphisms in some sheep breeds were observed. |
