Because of the need to improve the knowledge about canine perinatology, and given the major role of fetal fluids in sustaining the course of pregnancy and fetal development, an in-depth analysis to better understand the role of some hormones in these compartments is essential. Among all, leptin is recognized to play a key role not only on the energetic homeostasis, but also at multiple levels, influencing the control of reproduction, food assumption and metabolism. Even if in humans and other species it is reported the presence of leptin receptors during fetal development, very little is known about the canine species, in which the role of leptin still needs to be fully understood. The present study aimed to assess the amniotic fluid leptin (AFL) concentrations at term pregnancy in healthy dogs, and to evaluate the possible influence played by breed body-size (after assessment of correlation with maternal bodyweight and placental weight), or other maternal (age, parity, and the so-called "litter effect") and neonatal (gender, birth weight, litter size) parameters on AFL concentrations, analyzed by ELISA test. The study was performed on 90 healthy, viable and normal weighted puppies, 39 small-sized (adult body weight < 10 kg) and 51 large-sized (adult body weight > 25 kg), born by 29 purebred, healthy bitches, submitted to elective Caesarean section because of breed-related or individual high risk for dystocia. The results showed that the mean AFL concentration in the small-sized puppies was significantly (p < 0.05) higher in comparison to large-sized puppies (867.48 vs 698.42 pg/ml), while all the other studied parameters did not show to influence AFL concentrations. In conclusions, the present study showed significant higher at term AFL concentrations in small-sized as compared to large-sized breeds, suggesting an influence of breed body-size on fetal metabolism, as previously reported for NEFA and IGF-I.

A proper canine neonatal assistance, required to reduce the high perinatal loss rate, imply a full knowledge about the fetal-to-neonatal physiology. Because fetal fluids play an important role throughout mammals pregnancy, influencing fetal growth and development, fetal well being, and contributing to guarantee the most suitable environment for the fetus, the knowledge about fetal fluids biochemical composition is of major importance. At first, the biochemical composition of fetal fluids collected by normal developed, healthy and viable newborns, is necessary to depict the normal features, and represent the first step for the further detection of abnormalities associated to fetal/neonatal distress and useful for the early identification of newborns needing special attention, immediately after birth. The present study was aimed to define the biochemical composition of amniotic and allantoic fluids collected from fetus delivered by caesarean section at term of pregnancy. To reduce the possible confounding effect of maternal labor or troubles at parturition, fetal fluids were collected only from puppies born by elective caesaeran section, at term of normal pregnancies. Fetal fluids from 76 puppies, 70 normal and six pathologic newborns, born by elective caesarean section were collected and analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, lactate dehydrogenase (LDH), creatine-kinase (CK), alkaline phosphatase (ALP), creatinine, urea, amylase, lipase, gamma-glutamyl transferase (γ-GT), triglycerides, cholesterol, total proteins, albumin, globulins, glucose, magnesium, potassium, chloride, sodium, calcium, phosphorus and osmolarity. No significant differences were found between biochemical composition of amniotic or allantoic fluid in normal and pathologic newborns, maybe due to the small number of the pathologic puppies. Although some correlations between the two fluids were found (albumin, phosphorus, glucose and triglycerides), the results showed significant differences between the amniotic and allantoic biochemical composition (for all the parameters, except of alanine aminotransferase, triglycerides, cholesterol, albumin, amylase and glucose), suggesting that diverse sources could concur to the final composition of each fluid. A wide variability within and among litters was found for both amniotic and allantoic biochemical composition, and for some parameters an influence of breed body size (amniotic amylase, cholesterol, and allantoic calcium and glucose), maternal parity (amniotic and allantoic CK, glucose, LDH, chloride) and newborn gender (allantoic phosphorus) was found. Further investigations are needed for addressing the origin of each fetal fluid biochemical composition in the dog and also to indeep possible differences in fetal fluids biochemical composition between normal and pathologic puppies, providing potential markers for the quick identification of newborns that need special surveillance and cares immediately after birth.

Along the perinatal period, in mammals, cortisol (C) plays a pivotal role in the final intrauterine fetal maturation and in the early neonatal adaptation. Because of the scarce knowledge about canine perinatology, the present study was aimed to assess the C concentrations in amniotic and allantoic fluids collected, without invasiveness, from small-sized, purebred newborn puppies born by elective cesarean section, at term of pregnancy. Possible correlations between fetal fluid C concentrations and maternal parity, litter size, birth weight, Apgar score, were evaluated. In addition, the possible effect of fetal fluid C concentrations on newborn survival at 24 hours of age, and the effect of the litter or the newborn gender on fetal fluid C concentrations were also assessed. The results, obtained from 50 born alive, normal-weight puppies, without gross physical malformation, showed that C concentration was higher in allantoic than in amniotic fluid (P < 0.01), even if a strong positive correlation between the two fluids C concentration was found (P < 0.0001; R = 0.83). Neither amniotic nor allantoic C concentrations were correlated to maternal parity, litter size, birth weight, and Apgar score. Interestingly, higher amniotic (P < 0.05), but not allantoic, C concentrations were found in puppies not surviving at 24 hours after birth. Therefore, it could be suggested that this parameter may be useful for the recognition, at birth, of puppies needing special surveillance during the first day of age. A significant (P < 0.001) effect of the litter in both amniotic and allantoic C concentrations was found. In conclusion, the present results showed that in small-sized purebred puppies, born at term by elective cesarean section, the exact fetal, maternal, or placental source contributing to fetal fluid C concentrations remains to be clarified. From a clinical perspective, however, the evaluation of amniotic C concentration at birth seems useful for the detection of puppies that need special surveillance during the first 24 hours of age, and should be coupled to the early newborn evaluation by Apgar score. However, the small total number of newborns, and especially of the dead puppies enrolled in the present study, suggests that further, more-focused investigations on a large number of subjects are needed before the method could be considered for application in the clinical practices.

In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species.To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b).