محمدرضا جعفرزاده شیرازی

Benefiting from agricultural and garden residues is an efficient management tool to improve productivity in supplying protein needed by humans from animal sources (livestock and poultry). One of these residues is the waste resulting from harvesting and consumption of pomegranate fruits, one of the most important garden products in Iran. The peel and pomace of this fruit make up to 45% of its weight. Pomegranate is a fruit with high antioxidant properties that plays an important role in dealing with oxidative stress and can probably affect the hematological and biochemical blood parameters of living organisms. Therefore, this research aimed to investigate the blood parameters of old broilers fed with pomegranate peel to identify the possible adverse effects of this residue on hematological and biochemical parameters.

This research was conducted in the Faculty of Agriculture, Shiraz University, located 15 km northeast of Shiraz. Thirty-six 64-week-old roosters (Cob 500 strain) were assigned to three treatments in a completely random design, each with nine replicates of four birds. The experimental treatments included a T0 base diet without pomegranate peel and T10 and T20 diets with 10% and 20% pomegranate peel, respectively. To adapt to the experimental treatments, the roosters were fed experimental diets for 6 weeks, and blood samples were taken weekly from the brachial vein to examine blood hematological and biochemical parameters, including total number of white and red blood cells (WBC & RBC, resp.), WBC differential count (lymphocytes, monocytes, heterophils, and eosinophils), total protein concentration, albumin, total cholesterol, HDL (high-density lipoprotein), LDL (low-density lipoprotein), VLDL (very low-density lipoprotein), triglyceride, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase(AST), glucose, urea, and phosphorus. Data were analyzed with SAS software. This research was done in a completely randomized design, and the data were analyzed with a mixed procedure. The least squares means of the treatments, after correction for multiple comparisons based on Tukey's test, were compared at a significance level of 5%.

The main effect of experimental treatments was not significant on all the hematological parameters, and the main effect of time was significant only for monocytes (P < 0.05). The interaction of diet and time was significant only for the total number of RBCs (P < 0.05) and monocytes (P < 0.05). The main effect of the diet was decreasing on the concentrations of total cholesterol, LDL, and triglycerides while it was increasing on the concentration of HDL, glucose, alkaline phosphatase (ALP), and phosphorus. The effect of time was significant on the concentrations of total protein, total cholesterol, ALT, AST, and phosphorus. The interaction of diet and time significantly affected total cholesterol, ALP, AST, and phosphorus (P < 0.05). The analysis of variance showed the effect of diet, time, and the interaction of diet and time on the hematological parameters of Cobb 500 broilers fed with pomegranate peel powder. The effect of diet was not significant on all the hematological parameters. The effect of time was significant only on the percentage of monocytes (P < 0.05). The interaction of diet and time was significant on the total number of RBCs and the percentage of blood monocytes (P < 0.05). Weekly changes in the percentage of monocytes showed that the trend of changes during the experiment was increasing in the groups that received a high level of pomegranate peel powder (T20). From the second to the fifth weeks, the group in the T10 treatment showed a sharp decrease in the percentage of blood monocytes. Pomegranate peel powder feeding was associated with changes in blood concentrations of some biochemical parameters. Blood cholesterol concentration decreased in birds of the T20 and T10 groups. LDL and triglyceride concentrations decreased in birds fed with pomegranate peel powder at both feeding levels. In contrast, the concentrations of HDL, ALP, glucose, and phosphorus increased in these birds. The interaction effect of time and diet was also significant on the concentrations of cholesterol, ALP, phosphorus, and AST. Among the hematological parameters in birds fed with pomegranate peel powder, the percentage of monocytes was affected by time and the interaction of diet and time. Moreover, the total number of RBCs was affected by the interaction of diet and time.

Despite the significant differences in some of the mentioned parameters (monocytes, total number of RBCs, concentrations of total cholesterol, LDL, triglyceride, HDL, glucose, ALP, total protein, AIT, AST, and phosphorus) during the six-week period of pomegranate peel powder feeding in old broiler Cobb-500 chickens, the fluctuation range was in the normal physiological range. Therefore, pomegranate peel feeding at the tested levels (0, 10, and 20) was not associated with obvious damage to the general health and biochemical profile of birds. According to the findings of this research, it can be concluded that, although the nutrition of pomegranate peel powder could influence a number of blood parameters during the six-week feeding period, the fluctuation was within the normal physiological range despite the difference in the mentioned parameters. Therefore, pomegranate peel feeding at the levels used here was not associated with obvious damage to the general health and clinical profile of birds.

This research evaluated the blood and colostrum oxidative stress index (OSi) in newborn Holstein calves and its potential correlations with nutrient intake, growth performance, skeletal development, diarrhea, and pneumonia. Eighty-three neonatal Holstein calves were categorized based on their blood and consumed colostrum OSi levels. There were four treatment groups, i.e., 25 calves consumed low OSi colostrum that had low OSi in the blood taken 24-h after first meal consumption of colostrum (LL), 17 calves consumed low OSi colostrum that had high OSi in the blood (LH), 22 calves consumed high OSi colostrum that had low OSi in the blood (HL), and 19 calves consumed high OSi colostrum that had high OSi in the blood (HH). Upon categorization, the performance and health outcomes of the calves were thoroughly assessed. The results revealed no significant disparities among the treatment groups regarding nutrient intake and skeletal growth. Initial and final body weights, average daily gain (ADG), and feed efficiency had no observable differences. However, substantial differences were evident in the incidence of diarrhea (HH vs. LH, HL vs. LL, and LH vs. LL), pneumonia occurrence (HH vs. LH), and the number of days with body temperatures higher than 39.4 °C were similar across all groups. Moreover, a significant variation was noted in the duration of diarrhea, with the LL group experiencing more days under medication than the other treatment groups. Pronounced variations in calf health, particularly regarding diarrhea and pneumonia, suggesting a potential association between oxidative stress and specific health outcomes in newborn Holstein calves.

So far, changes in the vaginal microbial population during the estrous cycle have been evaluated in several livestock species such as sheep. However, changes in the rumen microbial population have not been investigated during the estrous period in sheep. In a study, the results showed that the differences in the rumen microbial population in two reproductive seasons were due to the differences caused by the reaction to day length, and nutrition did not have much effect on it. It is possible that during the reproductive and non-reproductive seasons, the animal has a different microbial population in the digestive tract. The available evidence from clinical research in animals shows that microbes in the digestive system can alter brain activity and behavior through hormonal and neural pathways. Therefore, this study mainly aims to investigate changes in the rumen microbial population during the reproductive season in the estrous period in ewes with estrus and those without estrus for any reason or are so-called anestrus. The changes in the rumen microbial population during the breeding season in the estrous period were measured for the animals showing the occurrence of estrus and those not showing this trait or were anestrous.

This research was done at the same time as the non-breeding season (late spring-beginning of July). The ewes were of Grey Shirazi breed (2-3 years old). To investigate changes in the rumen fluid microbial population in the estrus cycle in estrus and non-estrus animals, two groups of 10 animals were formed from estrus and anestrus animals with the use of teaser rams, and rumen fluid samples were collected for microbial culture and other investigations in both groups once per four days (5 times) during 17 days (like estrus cycle days). The colonies were cultured and separated using general culture media with different growth factors for anaerobic bacteria colonies, such as plate count agar, MRS agar (de Man-Rogosa - Sharpe), and starch agar. The colonies were subtracted by subtractive cultures or different diagnostic tests to identify colonies. Data were analyzed using SAS software version 1/9. Means were compared with the least square procedure and adjusted for Tukey's test.

The population difference between the two groups at 5 different times showed that the population of lactic acid bacteria (LAB) on the day of estrus showed a higher value and a significant difference with other times than the anestrus group. Lipolytic bacteria were higher on the day of estrus than on all days in both groups, with a significant difference. The comparison of different groups showed that the estrous group from times 1 to 5 in the breeding season had the highest number compared to the other groups at different times of the study (p < 0.05). Anestrous groups also had the lowest number at all times (p < 0.05). The comparison of the two estrus and anestrus groups showed that heat and ninth days had higher values than the other times. In comparing the average number of lipolytic colonies in the estrus group, the highest and the lowest numbers belonged to the 1st and the 4th times, respectively, which were significantly different from the other times (p < 0.05). The population of proteolytic bacteria had the highest number of bacteria at time 1 and showed the next values at times 2, 5, 3, and 4, respectively, compared to the estrus group at time 5 (p < 0.05). Time 5 was not significantly different from times 2 and 3 (p < 0.05). In this season, the estrus group was the highest and the lowest at times 1 and 4, respectively (p < 0.05). Times 3-5 were no different significantly (p < 0.05). In the anestrus group, times 3 and 1 had the highest number vs. the lowest numbers in times 2 and 5  (p < 0.05). Based on the comparison of the average population resulting from the counting of total bacterial colonies in the examination of different groups, the estrous group had the highest number of the average microbial population in all 5 times (p < 0.05). The lowest number belonged to two anestrus groups (p < 0.05). The population difference between the two groups at 5 different times showed that the population of LAB had a higher value only on the day of estrus, with a significant difference compared to the anestrous group. The population of lipolytic bacteria in two groups of estrus and anestrus in the 5 studied times showed that the estrus group had a higher and significant value than the anestrus group in the 4th time, i.e. the day of estrus and days 5, 6, and 17. The results showed that the total bacterial population was higher in the estrus group at all times, with a significant difference. Furthermore, anaerobic bacteria increased significantly in certain days of estrus (days 1-9) compared to the anestrous group. The population difference between the two groups at 5 different times indicated that the population of LAB had a higher value only on the day of estrus, with a significant difference compared to the anestrous group. The population of lipolytic bacteria in the two estrus and anestrus groups in the 5 studied times revealed that the estrus group had a higher and significant value than the anestrus group in 4 times, i.e. days of estrus, 5, 6, and 17. The total bacterial population in the estrous group was higher and significant at all times.

In general, the colony population of amylolytic and lipolytic bacteria and the total colonies of anaerobic bacteria are significantly different on days of the estrus cycle, which may affect reproduction. Accordingly, it is better to provide more understandable results of rumen microbial effects on estrus in ewes by increasing analyses such as hormonal changes and molecular investigations, along with microbial culture in estrus crops.

Changes in the microbial population of the digestive tract affect the reproductive and neoral systems. Both systems are involved in the process of estrus and ovulation. In this study, the main goal  wasis to investigate the changes in the rumen microbial population in non-breeding seasons during the estrous period for animals that have  signs of estrus and animals that do not show estrus signs.

In order to investigate changes in rumen fluid microbial population in estrus cycle, two groups of 10 animals in estrus and non estrus status were selected.  Once every four days rumen fluid samples were collected for microbial culture and other investigations from both groups. In order to cultivate and separate the colonies, general culture medium for anaerobic bacteria colonies such as Plate count agar, MRS agar (de Man-Rogosa - Sharpe) and starch agar was used. Subtraction of colonies was also done by subtractive cultures or different diagnostic tests to identify colonies. Data analysis was done using SAS software version 1/9. Means were compared with the least square procedure and adjusted for Tukey's test.

The  comparison between the two groups at 5 different times showed that only on the day of estrus, the population of lactic acid producing bacteria was higher than anestrous group (P<0.05). The comparison between two groups of estrus and anestrus  at 5 different times showed that the estrus group had a higher lipolytic bacterial population than the anestrus group in  days of  5, 6 and 17 of estrus cycle (P<0.05), but  at the day of 13th bacterial population in anestrus group  was higher (P<0.05). The comparison between estrus and anestrus groups showed that the amount of the total bacterial population in the estrus group at all the sampling times in the estrus group was higher than anestrus group (P<0.05).

In total, the cultures obtained from the rumen fluid in the estrus group showed that between amylolytic, lipolytic and total colonies of anaerobic bacterial populations in certain days of estrus cycle was different between animals with sign and no sign of estrus.

Affecting the female reproductive organ and the layers around the oocyst, some ration ions could facilitate the penetration of sperm with specific chromosome type and change the sex ratio in mammal infants. The purpose of the present study was to evaluate the effect of potassium supplement on the sex ratio in rat. The percentage of moisture, ash, crude fat and protein, sodium, potassium, calcium, and energy was measured in the ration of rats. In one gestation period, the male and female pups from 32 female and 8 male adult rats were counted as control group. In experimental group, potassium citrate (36% potassium) was used in control group ration to raise the level of potassium from 0.35% to 0.8% and 12 female and 3 male adult rats were fed with that ration in two gestation periods. The number of male and female pups in both groups was counted at the age of three days. Sex ratio in both groups was compared using chi-square test (SPSS, Ver. 11.5). The ration of control group 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 ratio of male pups in the rats were fed with potassium supplement (1.52, 100 male and 66 female pups) was higher (p=0.03) compared to the control group (0.94, 115 male and 122 female pups).CONLUSIONS: Raising the level of potassium in food ration can increase the number of male pups over 10% in each gestation.

Arginine-phenylalanine-amide-related peptide-3 (RFRP-3) is an inhibitor of gonadotropin releasing hormone (GnRH) secretion and an appetizer. Kisspeptin is a stimulator of GnRH and a regulator of energy metabolism. The objectives of the present study were to compare the effect of long term malnutrition on expression of RFRP-3 mRNA in dorsomedial hypothalamic nucleus (DMH) and KiSS-1 mRNA in arcuate nucleus (ARC) of rat hypothalamus. Sixteen female ovariectomized rats of the Sprague-Dawley strain were randomly allotted into three groups. After 2 weeks recovery period, one group was fed with perfect diet (rat standard feed) and the other group was fed with the half dietary of perfect diet for 14 days. The control group (n=4) were sacrificed 2 weeks after surgery (day 0). Relative expression of RFRP-3 and KiSS-1 mRNAs (compared to the control group) were measured using real-time PCR method in DMH and ARC of hypothalamus, respectively. Mean and SE of relative expression of RFRP-3 mRNA in DMH in the half dietary rats was higher than that of the perfect diet ones (P=0.01). Relative expression of KiSS-1 mRNA in ARC was not different between two groups of rats (P=0.1). Long term malnutrition (2 weeks) increased RFRP-3 mRNA expression in DMH of rats hypothalamus, but had no effect on KiSS-1 mRNA expression in the ARC nucleus of ovariectomized female rats.

In the present study number of neurons that express gonadotropin releasing hormone (GnRH) in the ewe preoptic area (POA) of hypothalamus were compared between the phases of estrous cycle. Hypothalamus of four ewe groups (n=3) of ovariectomized, proestrus, estrus, and luteal were removed after perfusion. The number of GnRH neurons in the POA of four groups was compared, using immunohistochemistry method. GnRH neurons were present in the POA of four groups of ewe. The mean of number of GnRH neurons in estrus ewes was more than ovariectomized ewes. However, there was not significant difference between the numbers of GnRH neurons of POA in three estrous phases. No difference for the number of neurons that express GnRH in the POA of hypothalamus during estrous phases of ewes might indicate that these relatively fixed number cells may perform the estrous cycle control only by control of GnRH release and changing of the secretion of GnRH.

Arginine-phenylalanine-amide-related peptide-3 (RFRP-3) and kisspeptin are believed to be inhibitor and stimulator of gonadotropin releasing hormone (GnRH) secretion, respectively. The aim of the present study was to compare the effect of lactation on the expression of RFRP-3 mRNA in dorsomedial hypothalamic nucleus (DMH) and KiSS-1 mRNA in arcuate nucleus (ARC) of hypothalamus in lactating and non-lactating rats. Fourteen rats of the Sprague-Dawley strain were randomly allocated into three groups. The non-lactating rats (n=5) were separated from their pups immediately upon parturition. The lactating rats were allowed to suckle five pups for eight days (the period of increasing of lactation). Relative expressions of RFRP-3 and KiSS-1 mRNAs compared to the control group were determined in DMH and ARC of hypothalamus, respectively, by using real-time PCR. Mean of relative expression of RFRP-3 mRNA in DMH in the lactating group was higher than that of the non-lactating rats (P=0.001). Relative expression of KiSS-1 mRNA in ARC did not show any difference between the lactating and the non-lactating rats (P=0.4). Increase of RFRP-3 mRNA expression in DMH of hypothalamus during the increase of milk production in rat may be the inhibitory factor for GnRH secretion. Whereas, no change in KiSS-1 mRNA expression in the ARC of the same rats may indicate the lower activity of this nucleus in the expression of the gene in both groups postpartum.

No domestic availability to breeder turkey stocks and turkey hatching eggs prompted the present experiment that aimed to evaluate the feasibility of a conventional artificial insemination (AI) procedure in British United Turkey (BUT) for the first time in Iran. Broiler turkeys were restrictedly fed, grown for 46 weeks, and used for the current study (10 turkey toms and 24 turkey hens in total). After a 3 week period of habituating the toms to abdominal massage, the pooled semen was used for insemination after the dilution in sterilized and homogenized low-fat milk (at the ratio of 1 to 6). The hens were inseminated (14:00 h) and hatching eggs were collected (n = 148). All the eggs were broken open to assess the fertility rate. Although being lower than the conventional average fertility rate noticed for breeder turkeys in the production manuals (91%), a fertility rate of 61.5% was obtained. The present report provided a preliminary data on the feasibility of the conventional procedure used in chickens to artificially inseminate the turkey, using low-fat milk as a simple available extender. The present findings might also be promising to the future establishment of turkey breeder enterprise in Iran.

Gonadotropin inhibitory hormone (GnIH) and Agouti-related peptide (AgRP) are orexigenic peptides expressed in the arcuate nucleus (Arc) of the hypothalamus in the ewe. In addition, effects of GnIH and AgRP on the regulation of gonadotropin releasing hormone secretion have been shown in some mammals. The objective of the present study was to investigate the coexpression of GnIH and AgRP in Arc of the ewe hypothalamus. Nine ewes were divided into follicular, luteal, and ovariectomised groups (n = 3 each group) and the number of neurons that express GnIH and AgRP and the percentage of coexpression of these two peptides in the Arc of each group was estimated by using immunohistochemistry. In different ovarian conditions of ewes, 19 to 32 percent of GnIH and AgRP neurons were coexpressed in the Arc of the hypothalamus. Results of the present study showed a possible cooperative role of AgRP and GnIH in the Arc of the hypothalamus in regulation of ovarian function in ewe.

Gonadotropin inhibitory hormone (GnIH) is believed to be an inhibitor of gonadotropin releasing hormone (GnRH) secretion. The role of GnIH in lactational inhibition of gonadotropin secretion is not well known; therefore, the aim of the present study was to evaluate the effect of lactation on expression of GnIH in these nuclei of lactating and non-lactating rats.

Ten postpartum rats of the Sprague-Dawley strain were randomly allotted into two groups. The number of pups was adjusted to five pups per suckling female, while the control non-lactating rats were separated from their pups immediately upon parturition. The lactating rats were allowed to suckle their pups for eight days. The number of GnIH neurons in the dorsomedial (DMH) and paraventricular (PVN) nuclei was estimated using an immunohistochemical method.

The number of GnIH neurons in DMH (P=0.005) and PVN (P=0.001) of lactating rats were significantly higher than those of the non-lactating rats. Moreover, fibers of GnIH neurons were observed in anteroventral periventricular nucleus (AVPV) in lactating rats.

Lactation increased GnIH expression in DMH and PVN of the lactating rats, and thus may have a role in inhibiting GnRH and subsequently LH secretion during the lactation period.

Agouti-related peptide (AgRP) and neuropeptide Y (NPY) are orexigenic peptideslocalized in arcuate nucleus (ARN) of the hypothalamus in ewe. Effects of NPY instimulation of luteinizing hormone (LH) secretion are stated in some mammals. The objective of the present study was to investigate level of AgRP expression in ARN of the hypothalamus in the estrus and diestrus phases in ewe. Six ewes were divided into estrous and diestrous groups (n=3) and the number of neurons that expressing the AgRP in the ARN of each group was estimated by using immunohistochemistry method. Without considering the different parts of ARN (rostral, middle, and caudal), the number of AgRP immunoreactive neurons in the estrus phase (63.55±14.37) was more than diestrus phase (31.22±6.34) (P=0.05). This finding indirectly showed that AgRP, may have an important stimulator role in the expression of LH and is reproductive axis in ewe.

Gonadotropin inhibitory hormone (GnIH) has been known as a key inhibitor of the secretion of gonadotropin releasing hormone (GnRH). Ewe has estrous cycles comprising of follicular and luteal phases. Follicular phase is in turn divided into proestrous and estrous phases. Blood level of LH increases in follicular and decreases in luteal phases. The aim of the present study was to evaluate (1) the presence of GnIH neurons in the ewe preoptic area (POA) and (2) the alterations of GnIH expression during different phases of ewe estrous cycle. Three fertile three-year-old ewes in each phase (n=9) were selected and the number of GnIH neurons was estimated by using immunohistochemistry method. GnIH neurons were present in the POA during different phases of estrous cycle. The number of GnIH neurons significantly increased in the luteal phase in comparison with the proestrous phase (P=0.001). GnIH expression in the neurons of POA of ewe is increased in the luteal phase compared to the follicular phase. This can inhibit GnRH secretion in POA and reduce LH secretion during the luteal phase.

The role of estrogen in the stimulation of gonadotropin-releasing hormone (GnRH) neurons is clear. These neurons do not express estrogen alpha receptors, so other mediator neurons should be present to transmit the positive feedback effect of estrogen to the GnRH neurons. Kisspeptin neurons have an important role in the stimulation of GnRH neurons, so they can be the mediator of the effect of estrogen on GnRH neurons in preoptic area of sheep brain. One of the known effects of estrogen is the stimulation of Fos gene in the brain. The aim of the present study was to determine the intermediary role of kisspeptin in the transmission of estrogen effects to the gonadotropin-releasing hormone neurons in the preoptic area of sheep brain. Six mature ewes in breeding season were selected and ovariectomised. Three ewes in treatment group were injected with 50 mg estradiol benzoate in 1 ml of sunflower oil and three ewes in control group were injected with saline solution intramuscularly. Immediately after estradiol injections, the hypothalamus of the ewes was removed. The count of kisspeptin neurons, Fos genes, and kisspeptin neurons which colocalized with Fos genes were determined by immunohistochemistry. Estradiol injection increased the colocalization of kisspeptin with Fos gene in the preoptic area of the sheep brain (P=0.01). Results of the present study showed that 86.9 percent of kisspeptin neurons colocalized with Fos gene in the preoptic area (P=0.01). Kisspeptin neurons are important mediators in transmission of positive feedback effect of estrogen into GnRH neurons in the preoptic area of sheep brain.