inhibition of estrous cycle in lactating rats mostly results from an inhibition of luteinizing hormone ( lh ) and gonadotropin releasing hormone ( gnrh ) secretion ( 2 ) .
suckling is an important inhibitory cue of lh surge during the first 8 days of lactation in rats ; separating pups from their dams restores lh secretion ( 3 ) .
furthermore , the administration of bromocriptine ( a dopamine agonist that is used in treatment of hyperprolactinemia ) has been shown to have no effect on the inhibitory effect of suckling on lh pulse ( 4 ) .
. suckling may be an appropriate model for studying the reproductive endocrine hormones involved during lactation .
kisspeptins belong to a family of peptides , encoded by the kiss-1 gene , mostly located in arcuate nucleus ( arc ) and anteroventral periventricular ( avpv ) region in rat brain ( 6 ) .
kisspeptin has a fundamental role in controlling the gonadal axis ( 7 ) , being a key regulator in lh secretion ( 8 , 9 ) .
kisspeptin neurons are located upstream of gnrh neurons and stimulate lh release by affecting these cells ( 10 ) .
administration of kisspeptin stimulated lh secretion and induced ovulation in female rats ( 11 , 12 ) . despite suppressed basal levels , lh secretory response to kisspeptin were preserved in pregnant and lactating female rats , although the magnitude of lh bursts and the sensitivity to kisspeptin were much higher in pregnant rats ( 13 ) .
alterations in kisspeptin and kiss-1 concentrations in the hypothalamus of rat during estrous cycle have been previously addressed ( 14 - 16 ) .
an increased kiss-1 mrna expression in arc probably mediates the negative feedback effect of estrogen on gonadotropin secretion , which might be the gnrh pulse generating center ( 15 ) .
in contrast to arc , kiss-1 mrna expression in avpv and preoptic nucleus was reduced following ovariectomy and increased through estradiol implantation .
it has been hypothesized that avpv might be involved in preovulatory gnrh / lh surge ( 15 ) .
the level of kiss-1 mrna in arc was highest during diestrus and lowest during proestrus in female rats ( 14 ) .
levels of lh pulsatile secretion are low in lactating rat ( 6 ) and human ( 18 ) .
estrus and ovulation are delayed about 20 days in lactating rats suckling 6 to 10 pups .
if the number of pups is greater than 12 with a 2-day - long water and food withdrawal period , then the strength of suckling is increased ( 19 ) .
therefore , the number of pups and strength of suckling may be critical stimulants in inhibiting the lh surge .
the aims of the present study were to evaluate the effects of litter size and intensity of suckling on the expression of kiss-1 mrna in lactating rats .
the findings would be beneficial to better clarifying the underlying mechanism(s ) involved in the lower reproductive performance due to lactation anestrous in high - producing dairy cows or in prolific species ( e.g. ovine and caprine ) . 

thirty two pregnant and 4 ovariectomized ( 3 - 4 months old ) female sprague - dawley rats ( rattus norvegicus ) , weighing 205.9  10.7 g ( mean  sd ) , were housed in individual cages under controlled temperature ( 22  2c ) and lighting ( 14 l : 10 d ; lighting starting at 07:00 through 21:00 ) with free access to food and water in the laboratory animal center of shiraz university of medical sciences , shiraz , iran .
the rats were fed 15 g / kg of food pellets diet which had 90% dry matter , 8% ash , 4.1% crude fat , 21.6% crude protein , 70.8% total digestible nutrients ( tdn ) , 0.4% calcium , 0.3% potassium and 0.1% sodium .
the rats were treated humanely and in compliance with the recommendations of the animal care committee of our college . 
 
the rats were randomly assigned into 9 groups ( n = 4 per group , table 1 ) .
the rats , assigned to the ovariectomized group , were anaesthetized by an intraperitoneal injection of ketamine ( 100 mg / kg ; woerden , netherlands ) and xylazine ( 7 mg / kg ; alfazyne , woerden , netherlands ) and ovariectomized through ventral midline incision .
lactating rats were allotted to 8 groups ( 4 rats each ) and they were allowed to suckle their pups until day 8 postpartum . in 3 groups ,
the litter size was adjusted to 5 , 10 , or 15 pups upon parturition and allowed to suckle their pups continuously . in further 3 groups of rats ,
the litter size was adjusted to 5 upon birth , the pups were separated from their dams on day 8 postpartum for 6 hr , after which the pups were allowed to suckle their dams for 2.5 , 5 , or 7.5 min before the dams were killed . the minimum of 2.5 min and 2.5 min intervals were selected according to the minimum time in increase in rna levels was immediately detected after transcription stimulation of a single cell ( 20 ) .
two groups of lactating rats with either 10 or 15 pups were similarly separated from their pups for 6 hr on day 8 postpartum , after which the pups were allowed to suckle their dams for 5 min , before the dams were killed . 
 
rats were anaesthetized by ether and killed via cervical dislocation at 15:00 to 16:00 on day 8 postpartum .
brains were immediately removed and the diencephalon was dissected out by an anterior coronal section , anterior to the optic chiasm , and a posterior coronal cut at the posterior border of the mammillary bodies . to separate arc , a third coronal cut was made through the middle of the optic tract , just rostral to infundibulum ( 21 ) .
total rna was extracted , using the rnx - plus buffer ( cinnagen , tehran , iran ) .
briefly , the tissue ( 100 mg ) was ground in liquid nitrogen , transferred to rnx - plus buffer ( 1 ml ) in an rnase - free microtube , mixed thoroughly , and kept at room temperature for 5 min .
the mixture was centrifuged at 12,000  g ( 4c ) for 20 min and the supernatant was transferred to another tube and precipitated with an equal volume of isopropanol for 15 min .
the rna pellet was washed with 75% ethanol and quickly dried and re - suspended in 50 l rnase - free water . the purified total rna was quantified by nano - drop nd 1000 spectrophotometer ( nano - drop techno - logies , wilmington , de , usa ) .
the dnase - treated rna ( 3 g ) was used expression of the target mrnas over the reference for the first strand cdna synthesis , using 100 pmol oligo - dt , 15 pmol dntps , 20 u rnase inhibitor , and 200 u m - mulv reverse transcriptase ( fermentas , st .
primers were designed , using allele i d 7 software ( premier biosoft international , palo alto , usa ) for reference gene and kiss-1 ( nm_181692 ) .
the rat glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene ( m32599 ) was used as a reference gene for data normalization ( table 2 ) .
relative real - time pcr was performed in a 20 l volume containing 1 l cdna , 1x syber green buffer and 4 pmol of primer .
the amplification reactions were carried out in a line - gene k thermal cycler ( bioer technology co. , ltd , hangzhou , china ) under the following conditions : 2 min at 94c , 40 cycles of 94c 10 sec , 57c 15 sec , and 72c 30 sec .
after 40 cycles , the specificity of the amplifications was tested by heating from 50c to 95c , resulting in melting curves .
all amplification reactions were repeated 3 times under identical conditions , including a negative control and 5 standard samples . to ensure that the pcr products were generated from cdna , but not the genomic dna , proper control reactions were implemented in the absence of reverse transcriptase . for quantitative real - time pcr data , the relative expression of the kiss-1
the ct for each sample was calculated , using line - gene k software ( 22 ) .
accordingly , the fold values was calculated by the equation 2(23 ) , where ct is determined by subtracting the corresponding gapdh ct value ( internal control ) from the specific ct of the target ( kiss-1 ) .
the ct was obtained by subtracting the ct of each experimental sample from that of the calibrator one ( non - lactating ovariectomized rats ) . 
 
the data on relative expression of kiss-1 gene were subjected to the test of normality and analyzed by one - way anova ( sas 9.1sas institute inc . , cary , nc ) , and mean separation was performed by tukey 's test at p = 0.01 . 

thirty two pregnant and 4 ovariectomized ( 3 - 4 months old ) female sprague - dawley rats ( rattus norvegicus ) , weighing 205.9  10.7 g ( mean  sd ) , were housed in individual cages under controlled temperature ( 22  2c ) and lighting ( 14 l : 10 d ; lighting starting at 07:00 through 21:00 ) with free access to food and water in the laboratory animal center of shiraz university of medical sciences , shiraz , iran .
the rats were fed 15 g / kg of food pellets diet which had 90% dry matter , 8% ash , 4.1% crude fat , 21.6% crude protein , 70.8% total digestible nutrients ( tdn ) , 0.4% calcium , 0.3% potassium and 0.1% sodium .
the rats were treated humanely and in compliance with the recommendations of the animal care committee of our college . 

the rats were randomly assigned into 9 groups ( n = 4 per group , table 1 ) .
the rats , assigned to the ovariectomized group , were anaesthetized by an intraperitoneal injection of ketamine ( 100 mg / kg ; woerden , netherlands ) and xylazine ( 7 mg / kg ; alfazyne , woerden , netherlands ) and ovariectomized through ventral midline incision .
lactating rats were allotted to 8 groups ( 4 rats each ) and they were allowed to suckle their pups until day 8 postpartum . in 3 groups ,
the litter size was adjusted to 5 , 10 , or 15 pups upon parturition and allowed to suckle their pups continuously . in further 3 groups of rats ,
the litter size was adjusted to 5 upon birth , the pups were separated from their dams on day 8 postpartum for 6 hr , after which the pups were allowed to suckle their dams for 2.5 , 5 , or 7.5 min before the dams were killed . the minimum of 2.5 min and 2.5 min intervals were selected according to the minimum time in increase in rna levels was immediately detected after transcription stimulation of a single cell ( 20 ) .
two groups of lactating rats with either 10 or 15 pups were similarly separated from their pups for 6 hr on day 8 postpartum , after which the pups were allowed to suckle their dams for 5 min , before the dams were killed . 

rats were anaesthetized by ether and killed via cervical dislocation at 15:00 to 16:00 on day 8 postpartum .
brains were immediately removed and the diencephalon was dissected out by an anterior coronal section , anterior to the optic chiasm , and a posterior coronal cut at the posterior border of the mammillary bodies . to separate arc , a third coronal cut was made through the middle of the optic tract , just rostral to infundibulum ( 21 ) .
total rna was extracted , using the rnx - plus buffer ( cinnagen , tehran , iran ) .
briefly , the tissue ( 100 mg ) was ground in liquid nitrogen , transferred to rnx - plus buffer ( 1 ml ) in an rnase - free microtube , mixed thoroughly , and kept at room temperature for 5 min .
the mixture was centrifuged at 12,000  g ( 4c ) for 20 min and the supernatant was transferred to another tube and precipitated with an equal volume of isopropanol for 15 min .
the rna pellet was washed with 75% ethanol and quickly dried and re - suspended in 50 l rnase - free water . the purified total rna was quantified by nano - drop nd 1000 spectrophotometer ( nano - drop techno - logies , wilmington , de , usa ) .
the dnase - treated rna ( 3 g ) was used expression of the target mrnas over the reference for the first strand cdna synthesis , using 100 pmol oligo - dt , 15 pmol dntps , 20 u rnase inhibitor , and 200 u m - mulv reverse transcriptase ( fermentas , st .
primers were designed , using allele i d 7 software ( premier biosoft international , palo alto , usa ) for reference gene and kiss-1 ( nm_181692 ) .
the rat glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene ( m32599 ) was used as a reference gene for data normalization ( table 2 ) .
relative real - time pcr was performed in a 20 l volume containing 1 l cdna , 1x syber green buffer and 4 pmol of primer .
the amplification reactions were carried out in a line - gene k thermal cycler ( bioer technology co. , ltd , hangzhou , china ) under the following conditions : 2 min at 94c , 40 cycles of 94c 10 sec , 57c 15 sec , and 72c 30 sec .
after 40 cycles , the specificity of the amplifications was tested by heating from 50c to 95c , resulting in melting curves .
all amplification reactions were repeated 3 times under identical conditions , including a negative control and 5 standard samples . to ensure that the pcr products were generated from cdna , but not the genomic dna , proper control reactions were implemented in the absence of reverse transcriptase . for quantitative real - time pcr data , the relative expression of the kiss-1
the ct for each sample was calculated , using line - gene k software ( 22 ) .
accordingly , the fold values was calculated by the equation 2(23 ) , where ct is determined by subtracting the corresponding gapdh ct value ( internal control ) from the specific ct of the target ( kiss-1 ) .
the ct was obtained by subtracting the ct of each experimental sample from that of the calibrator one ( non - lactating ovariectomized rats ) . 

the data on relative expression of kiss-1 gene were subjected to the test of normality and analyzed by one - way anova ( sas 9.1sas institute inc . , cary , nc ) , and mean separation was performed by tukey 's test at p = 0.01 . 

relative expression of kiss-1 gene in rats continuously housed with 5 pups was greater than those suckling either 10 or 15 pups ( p < 0.0001 ; figure 1 ) .
relative expression of kiss-1 gene in the arc of lactating rats with 5 pups which were separated from their pups for 6 hr but allowed suckling for 2.5 min was greater than in rats suckled for 5 or 7.5 min ( p < 0.0001 ; figure 2 ) .
relative expression of kiss-1 gene in the arc of rats suckled for 2.5 min was lower than in rats continuously housed with the same number of pups ( p = 0.0002 ) .
relative expression of kiss-1 gene in the arc of lactating rats continuously housed 5 , 10 , or 15 pups were respectively greater ( p < 0.0001 ) than in rats separated from their pups but allowed suckling for 5 min following 6 hr removal of the pups ( figure 3 ) . there were no differences in the relative expression of kiss-1 gene in the arc of lactating rats with 5 , 10 , and 15 pups which were separated from their pups and the rats which were suckled for 5 min after 6 hr removal of pups ( p > 0.05 ) . 

expression of kiss-1 mrna in the arc of lactating rats was decreased as the number of suckling pups was increased .
consistent with our findings , yamada et al(17 ) reported that suckling stimulus inhibited the expression of kisspeptin in arc neurons .
increased number of pups per lactating rat results in higher milk production , and therefore the negative energy balance becomes more exacerbated ( 24 ) .
regardless of the level of energy intake , the efficiency of energy use is increased substantially during lactation in rats .
the mechanisms involved in negative energy balance play an important role in the change ( 24 ) .
kiss-1 neurons are direct targets for regulation by leptin(25 ) and leptin levels were low in underfed rats ( 26 ) .
decreased leptin production and action during lactation increases feed intake , necessary for successful rearing of the offspring ( 27 ) . during periods of negative energy balance ,
have no receptor for leptin(29 ) . therefore , during early lactation , low leptin levels may suppress kisspeptin and inhibit gnrh neuron .
the present study showed that increased intensity of suckling resulted in lower expression of kiss-1 mrna , with a greater influence of increased suckling intensity compared to the litter size .
there is little data reporting the relationship between lactation stress and expression of kisspeptin , but it has been shown that corticotrophin - releasing factor decreased kiss-1 expression in arc of female rats ( 30 )
. there are projections from corticotrophin - releasing factor neurons in the paraventricular nucleus to the regions where kisspeptin neurons are found ( 30 ) .
an inverse relationship was reported between the blood levels of adrenocorticotropin and corticosterone during lactation in rat ( 31 ) . moreover , cortisol treatment suppressed lh release in rats ( 32 ) .
suckling is also an important inhibitor of lh surge in lactating rats ( 3 ) .
therefore , increased glucocorticoid secretion due to increased litter size and/or suckling intensity may inhibit gnrh secretion through suppression of kisspeptin .
results of the present study showed the negative effect of litter size on the expression of kiss-1 gene .
a relationship was reported between the height of the suckling - induced prolactin ( prl ) rise and litter size in rat ( 33 ) .
there was a high degree of colocalization of prl receptor mrna in kiss-1 mrna - containing cells ( 34 ) .
moreover , tuberoinfundibular dopaminergic ( tida ) neurons in arc are known as the key regulator of prl release ( 35 ) .
there is a close apposition between kisspeptin fibers and tida neurons in the arc , suggesting kisspeptin may contribute to prl release ( 35 ) .
decreased kisspeptin was conversely correlated with lh secretion , and prl inhibited lh secretion ( 36 ) .
one hypothesis is that kisspeptin neurons are inhibited as a result of suckling , and that some of kisspeptin fibers affect tida neurons and prl secretion , with some kisspeptin neurons projecting to poa to stimulate gnrh release .
expression of neurokinin b ( nkb ) in arc neurons were decreased in lactating rats and nkb neurons colocalise with kisspeptin neurons ( 37 ) .
nkb neurons in arc are thought to stimulate lh release ( 37 ) , therefore , the suckling stimulus may inhibit kisspeptin and nkb neurons . 

an increased litter size and intensity of suckling decreased kiss-1 mrna expression in rat hypothalamic arc which may suppress the pulsatile gnrh / lh secretion during lactation anestrus .
the findings of the present study may help to characterize the mechanism(s ) contributing to a decreased reproductive performance in high - producing dairy cows or in those twinning is a common phenomenon ( e.g. ovine and caprine ) . 

the results described in this paper were part of student thesis and this research was financially supported by the shiraz university vice - chancellor for research , shiraz university , shiraz , iran . 

   objective(s):the effect of litter size and suckling intensity on the expression of kiss-1 mrna in the arcuate nucleus ( arc ) of rats were evaluated.materials and methods : thirty two pregnant and four non - lactating ovariectomized ( as control group ) rats were used in this experiment . 
 lactating rats were allotted to eight equal groups . in three groups , 
 litter size was adjusted to 5 , 10 , or 15 pups upon parturition and allowed to suckle their pups continuously by 8 days postpartum . in the other three groups , 
 litter size was adjusted to five upon birth ; the pups were separated from the dams for 6 hr on day 8 postpartum , after which the pups were allowed to suckle their dams for 2.5 , 5 , or 7.5 min prior to killing the dams . 
 two groups of lactating rats with either 10 or 15 pups were separated from their pups for 6 hr on day 8 postpartum , after which the pups were allowed to suckle their dams for 5 min before the dams were killed on day 8 postpartum . 
 the arc was removed and the expression of kiss-1 mrna was evaluated , using real - time pcr.results : the expression of kiss-1 mrna in the arc was decreased as the litter size and intensity of suckling stimulus were increased . 
 the effect of suckling intensity on the expression of kiss-1 mrna was more pronounced than that of litter size.conclusion:increased litter size and suckling intensity decreased kiss-1 mrna expression in the arc which may contribute to lactation anestrus in rat . 
Introduction
Materials and Methods
Animals
Experimental groups
Sampling
Real-time PCR
Statistical analysis
Results
Discussion
Conclusion
Acknowledgment
Conflict of interest