Non-invasive assessment of hormonal fluctuations during pregnancy in guanacos (Lama guanicoe) and its application in a wild population.

Obtaining endocrinological profiles using non-invasive methodologies by the measurement of hormone fecal metabolites is a widely used method to monitor ovarian activity and pregnancy in wild species. These tools allow the obtention of physiological information without causing capture-related stress on the individuals. In this research, we aimed to 1) biologically validate a non-invasive method to assess fecal progestagens and estrogens fluctuations during gestation in guanacos (Lama guanicoe) and 2) apply this technique to assess pregnancy in a wild free-ranging population. Fecal samples were collected through the gestation period (~12 months) of female guanacos in a 6.5-ha paddock. An increase in fecal metabolites of both hormones was detected. Progestagens increased gradually, in contrast to estrogens, which remained at basal values for most of the gestation period and peaked only a few days before calving. To assess pregnancy in wild free-ranging animals, fecal samples were collected from a population of La Payunia provincial reserve (Mendoza, Argentina) during the beginning of gestation and at the end of gestation. Through the first months of possible gestation, pregnant females represented between 40 and 80% of the population; at the end of gestation, only 20-40% of the females had confirmed pregnancies. Our results demonstrated that the polyclonal antisera and sexual hormone metabolite assays used here detect variations in the metabolites excreted through feces in guanacos and provide the possibility of non-invasive hormone monitoring of female reproductive status. Also, the findings in wild conditions suggest that natural abortions could have occurred during the first months of gestation. Although some abortions may be natural, the harsh environmental conditions that challenge the support of such a long gestational process may be another relevant factor to consider. The results obtained here enhance our understanding of the reproductive physiology of one of the most emblematic ungulates in South America.

The role of intragestational ghrelin on postnatal development and reproductive programming in mice

The purpose of this study was to evaluate the intragestational role of ghrelin in offspring development and reproductive programming in a mouse model of ghrelin imbalance during pregnancy. Female mice were injected with ghrelin (supraphysiological levels: 4 nmol/animal/day), antagonist (endogenous ghrelin inhibition with (D-Lys3)GHRP-6, 6 nmol/animal/day) or vehicle (control = normal ghrelin levels) throughout the pregnancy. Parameters evaluated in litters were growth, physical, neurobiological and sexual development and, at adulthood, reproductive function. Litter size and initial weight did not vary between treatments. Male pups from dams treated with ghrelin showed higher body weight increase until adulthood (31.7 ± 0.8 vs control = 29.7 ± 0.7, n = 11­14 litters/treatment; P < 0.05). Postnatal physical and neurobiological development was not modified by treatments. The antagonist accelerated male puberty onset, evidenced as earlier testis descent and increased relative testicular weight (antagonist = 0.5 ± 0.0% vs ghrelin = 0.4 ± 0.0% and control = 0.4 ± 0.0%, n = 5­10 litters/treatment; P < 0.05). At adulthood, these males exhibited lower relative testicular weight and reduced sperm motility (63.9 ± 3.6% vs control = 70.9 ± 3.3 and ghrelin = 75.6 ± 3.0, n = 13­15 animals; P < 0.05), without changes in plasma testosterone or fertility. Female pups intragestationally exposed to the antagonist showed earlier vaginal opening (statistically significant only at Day 25) and higher ovarian volume (antagonist = 1085.7 ± 64.0 mm3 vs ghrelin = 663.3 ± 102.8 mm3 and control = 512.3 ± 116.4 mm3; n = 4­6 animals/treatment; P < 0.05), indicating earlier sexual maturation. At adulthood, these females and those exposed to ghrelin showed a tendency to higher percentages of embryo loss and/or foetal atrophy. In conclusion, ghrelin participates in reproductive foetal programming: alterations in ghrelin activity during pregnancy modified body weight increase and anticipated puberty onset, exerting (or tending to) negative effects on adult reproductive function.

Maternal and postnatal high-fat diets with high ω6 : ω3 ratios affect the reproductive performance of male offspring in the mouse.

High-fat diets (HFDs) are an acknowledged risk factor for male subfertility, but the underlying mechanisms remain unclear. In the present study we compared the effects of two HFDs with different ω6:ω3 ratios, one enriched with soy oil (SOD; ω6:ω3=9.62) and another enriched with sunflower oil (SFOD; ω6:ω3=51.55), with those of a commercial diet (CD; ω6:ω3=19.87), supplied from pregnancy to adulthood, on morphometric parameters and reproductive performance in adult male mice (recommended ω6:ω3 for rodents=1-6). Bodyweight was significantly higher in the SFOD than CD group, and relative testicular weight was significantly lower in the SFOD than the other two groups. SFOD altered sperm performance: it reduced sperm viability (mean±s.e.m.; 76.00±1.35% vs 82.50±1.45% and 80.63±1.00% in the SFOD vs CD and SOD groups respectively; P<0.05) and increased the percentage of immature spermatozoa (71.88±7.17% vs 51.38±5.87% and 48.00±5.72% in the SFOD vs CD and SOD groups respectively; P<0.05). The epididymal ω6:ω3 ratio was higher in the SFOD versus CD and SOD groups, whereas the unsaturation index was higher in the SOD and SFOD groups than in CD group. Sperm membrane integrity was diminished in both the SOD and SFOD groups, but there was no difference in sperm reactive oxygen species production in these two groups compared with the CD group. The fertilisation rate was lower in the SFOD compared with the CD and SOD groups. In conclusion, although both HFDs affected sperm quality, the fertilising ability was more altered by the excessive dietary ω6:ω3 ratio than by the net ω6 content.

Subclinical lower airways correlates of chronic allergic and non-allergic rhinitis.

The upper and lower airways behave as a physiological and pathophysiological unit. Subclinical lower airways abnormalities have been described in patients with rhinitis without asthma. These are expressed as bronchial hyperreactivity, abnormalities in lung function and bronchial inflammation, likely as a result of the same phenomenon with systemic inflammatory impact that reaches both the nose and the lungs, which for unknown reasons does not always have a full clinical expression. Patients with rhinitis are at increased risk of developing asthma; therefore, most authors suggest a careful clinical evaluation and monitoring of these patients, especially if symptoms related to inflammation in the lower airways are observed. Although current treatments, such as H1-antihistamines, intranasal steroids and allergen immunotherapy, are quite effective for the management of rhinitis, it is difficult to prove their capacity to prevent asthma among subjects with rhinitis. Evidence showing that the treatment of rhinitis has a favourable impact on indicators of bronchial hyperreactivity and inflammation among subjects that have no symptoms of asthma is more frequently described. In this review, we address the frequency and characteristics of lower airway abnormalities in subjects with rhinitis, both in paediatric and adult populations, their likely predictive value for the development of asthma and the possibilities for therapeutic intervention that could modify the risk of subjects with rhinitis towards presenting asthma.

Impact of rhinitis on lung function in children and adolescents without asthma.

BACKGROUND: Subclinical spirometric abnormalities may be detected in patients with rhinitis without asthma, proportional to the severity established by ARIA (Allergic Rhinitis and Its Impact on Asthma) guidelines. New criteria of rhinitis classification were recently validated according to the ARIA modified (m-ARIA), which allow the discrimination between moderate to severe grades. The impact of rhinitis on lung function according to frequency and severity is unknown. OBJECTIVES: To investigate subclinical spirometric impairment in children and adolescents with allergic and non-allergic rhinitis without overt symptoms of asthma, according to the frequency and severity criteria of rhinitis classified by m-ARIA. METHODS: An observational cross-sectional study, including children and adolescents aged 5-18 years with allergic and non-allergic rhinitis without asthma. We analysed the functional abnormalities and bronchodilator response with spirometry in relation to the grade of rhinitis established by m-ARIA using an adjusted logistic model. A value of p<0.05 was considered statistically significant. RESULTS: We studied 189 patients; 22.2% showed spirometric abnormalities. Patients with persistent rhinitis had greater impairment of lung function compared to intermittent grade (p=0.026). Lung functional impairment was more frequent in severe and moderate rhinitis than mild grade (p=0.005) and was independent of the atopic status to both frequency (p=0.157) and severity (p=0.538). There was no difference in bronchodilator reversibility between groups (p>0.05). CONCLUSIONS: Impaired lung function was associated with persistence and severity of rhinitis and there was no significant difference between patients with moderate and severe rhinitis. The spirometric abnormality was demonstrated in patients with allergic and non-allergic rhinitis.

Seasonal evaluations of urinary androgen metabolites and semen quality in domestic long-tailed chinchilla (Chinchilla lanigera) under natural photoperiod.

Chinchilla spp. is a South American hystricomorph rodent genus currently considered almost extinct in the wild. The high quality of chinchilla fur motivated the harvesting of chinchillas for the fur market. Reproductive biology advances come from studies on commercially exploited animals, especially Chinchilla lanigera. We studied seasonal variation of urinary androgen metabolites, sperm concentration and sperm functional activity in males of domestic Chinchilla lanigera under natural photoperiod. In Córdoba city (31° S-64° W; Argentina), within the same latitudes as those of the historic Andean distribution (tropical deserts; 15°-34° S), domestic males (n=7) were studied in May (autumn), August (winter), November (spring), and February (summer). Urine was seasonally collected (over 24h; once for season, 4 in total) to measure urinary androgen metabolites (RIA), before semen collection by electroejaculation. The results indicated that although testicular volume (relative to body weight) and values of sperm functional activity did not show seasonal changes, a seasonal variation in androgen excretion was detected, with the highest values occurring during "short" light/dark cycles (autumn-winter). In addition, viable spermatozoa with intact acrosome mean values during winter-spring were higher than in autumn or summer. This study provides information that might contribute to the assessment of testicular activity in male chinchilla subjected to genetic selection in the fur industry. In addition, since domestic chinchilla still share some genomic characteristics with their counterparts in the wild, results presented may alsocontribute to ex situ breeding program of endangered chinchilla. In conclusion, natural photoperiod cycle affects testicular activity in domestic chinchilla.

Mouse plasma progesterone levels are affected by different dietary ω6/ω3 ratios.

An imbalance in the dietary polyunsaturated fatty acids (PUFAs) ω6/ω3 ratio, could influence negatively the reproductive performance. The aim of the study was to assess the effects of chronic administration of diets enriched with soybean or sunflower oils with different ω6/ω3 ratios on the reproductive parameters of adult female mice. Mice were fed different diets for 90 days: a commercial diet (CD), a 5 or 10% soy oil-enriched diet (SOD5 and SOD10, respectively), and a 5 or 10% sunflower oil-enriched diet (SFOD5 and SFOD10, respectively). The parameters evaluated were: body weight and food intake, estrous cycle, plasma progesterone concentration, ovulation rate, and oocyte quality. Progesterone concentrations (ng/ml) were significantly higher in the SFOD10: 14.9±2.8 vs CD: 5.4±1.2; SOD5: 5.6±1.1 and SFOD5: 4.6±1.4. Additional parameters evaluated were not affected. However, metestrous and luteal phases were shorter in subjects receiving SOD and longer in those under SFOD diets. In SFOD, there was a trend towards a smaller number of recruited oocytes compared to CD and SOD and a higher percentage of cleaved oocytes were quantified in SOD diets. A 3-month supply of a diet with elevated LA ω6/ALA ω3 ratio to adult female mice affects their reproductive physiology, modifying progesterone production, ovulation rate, and/or oocyte quality. Although some differences in the response to diets have been observed in several mammalian species, the present findings must be taken into consideration when a diet for optimizing reproductive capability is indicated.

Reproduction in chinchilla (Chinchilla lanigera): current status of environmental control of gonadal activity and advances in reproductive techniques.

A review of the biology of reproduction of chinchilla, focusing on environmental control of the gonadal activity, is presented. Chinchilla is a South American hystricomorph rodent genus currently considered almost extinct in the wild. However, a domestic form is still widespread in breeding farms around the world. Information regarding their reproductive biology has been obtained from studies on captive animals. In the case of Chinchilla lanigera, a seasonal reproductive pattern has been frequently reported in breeding facilities, but factors that might trigger gonadal activity have not been identified. The available information on reproductive productivity in farms worldwide shows a range of 1.2 to 2.4 deliveries per female per yr (with up to 2.1 weaned young per female per yr). Indeed, as found in all rodents, chinchillas can multiply at high fecundity and fertility rates (4 to 6 follicles mature during estrous cycles). Some new research avenues are postulated to improve the control of gonadal activity by means of environmental and/or pharmacologic factors. Furthermore, reproductive techniques that have been validated in chinchilla are reviewed (noninvasive hormone monitoring, semen collection, sperm cryopreservation, estrus induction), and several technical steps are proposed to be able to achieve AI. Because domesticated chinchilla still share some genomic characteristics with their counterparts in the wild, validated reproductive techniques in chinchilla males and females might contribute to the success of breeding programs.

Seminal quality and neutral alpha-glucosidase activity after sequential electroejaculation of chinchilla (Ch. lanigera).

The objectives of this study of seminal quality of chinchilla (Chinchilla lanigera) males were to evaluate (1) the use of neutral alpha-glucosidase (NAG) as a functional epididymal marker in this species, and (2) the effects of repetitive electroejaculation on sperm function and seminal NAG concentration. Semen was obtained by electroejaculation from sexually mature domestic Ch. lanigera males (n=6) once a week for six consecutive weeks, and evaluated to assess seminal volume and NAG activity, and sperm concentration and functional activity (motility, viability, membrane and acrosome integrity). Body weight was determined in all animals before each electroejaculation procedure. NAG activity was detected in all semen samples obtained, reaching a concentration of 55.4±6.8µU/mL. No significant variations were observed throughout the experimental period in any of the semen quality parameters evaluated. A reduction (P=0.001) in body weight was detected from the third week of experimentation and by the end of the experiments the animals lost a 7.2±0.9% of body weight. Overall, results in the present study confirm that NAG activity is detectable in chinchilla seminal plasma and that repetitive electroejaculation is not deleterious, on a short-term basis, to the semen characteristics.

Functional activity of frozen thawed Chinchilla lanigera spermatozoa cryopreserved with glycerol or ethylene glycol.

The cryopreservation of spermatozoa constitutes a valuable tool for the captive breeding management of valuable and/or threatened species. Chinchilla lanigera is a species almost extinct in the wild, and the domestic counterpart has one of the most valuable pelts in the world. The objectives of this study were to: (i) compare the functional activity of post-thawed chinchilla spermatozoa cryopreserved at -196 degrees C either with glycerol (G) or ethylene glycol (EG) as cryoprotectants (1 m final concentration) and (ii) investigate the effects of incubating the gametes for 4 h in the presence or in the absence of the cryoprotectants; evaluations were performed taking into account motility, viability, response to hypo-osmotic shock and acrosome integrity of the cells. Parameters reflecting post-thaw (0 h) sperm functional activity were significantly lower than those of freshly ejaculated gametes. When comparing the cryoprotectant efficiency of G vs EG, neither cryoprotectant agent offered appreciable advantages. After 4 h of incubation, in the presence or absence of the cryoprotectant agent, a rapid and significant decrease was found in all functional parameters and remained at approximately 20-30% motile, viable and viable acrosome intact cells. Viability was significantly lower when the cryoprotectant was removed from the media (possibly due to the centrifugation process). With respect to the maintenance of sperm membrane integrity, only approximately 10% of cells showed membrane resistance to hypo-osmotic conditions after the 4 h incubation period. These results constitute new insights for cryopreservation protocols and the development of assisted reproductive techniques in this species.

Electroejaculation in the Chinchilla (Chinchilla lanigera): effects of anesthesia on seminal characteristics.

Repetitive electroejaculation is probably extremely stressful in conscious animals and could adversely affect fertility. The present study was designed to (a) evaluate the effects of anesthesia (40 mg ketamine/kg body weight, i.m.) on a method of electroejaculation used previously in conscious chinchillas (Chinchilla lanigera), and (b) determine the quality of the electroejaculated semen obtained under anesthesia. In Experiment 1 (8 animals), a 4 x 4 Latin square design was used to study the effects of anesthesia and ejaculatory voltage on semen collection, ejaculate volume, sperm concentration, motility, viability, response to the hypoosmotic swelling test (HOST), and acrosomal status. In Experiment 2 (12 animals), the effects of differing voltages and the number of stimuli on ejaculation by conscious or anesthetized males was determined. In both experiments, all the conscious animals ejaculated, but only 60% ejaculated under anesthesia and they required more stimuli and higher voltages to achieve ejaculation. Ejaculate volume was significantly lower in anesthetized (<5 microl) than in conscious animals (>40 microl), but sperm concentration was unaffected. None of the indices of sperm quality were affected by anesthesia. The techniques we have developed in anesthetized domestic chinchillas should be applicable to endangered chinchillas in the wild, and though the number of sperm available is reduced, there are still sufficient for assisted reproduction.

Storage of Chinchilla lanigera semen at 4 degrees C for 24 or 72 h with two different cryoprotectants.

The objectives of this study were to: (a) test the functional activity of Chinchilla lanigera spermatozoa suspended in either glycerol or ethylene glycol, cooled to 4 degrees C, and stored for 24 or 72 h and (b) investigate, after these cooling periods, the effects of incubating sperm at 37 degrees C (for 4 h) upon sperm functional activity. The ejaculate was mixed with the cryoprotectant medium (at 1 M final concentration) and cooled to 4 degrees C. After warming, sperm motility, sperm viability, hypoosmotic swelling test results, and acrosomal integrity were significantly higher for samples containing ethylene glycol than for those in glycerol, stored for 24 or 72 h, and then assayed after 0 or 4 h incubation at 37 degrees C. A significant reduction of sperm motility and viability was detected only when the glycerol cryoprotectant agent was employed, compared to the fresh samples. These results clearly indicate that under our experimental conditions, ethylene glycol is a better protectant for sperm storage than glycerol.

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved.

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.