F. R. C. L. Almeida

Intra-uterine growth retardation affects birthweight and postnatal development in pigs, impairing muscle accretion, duodenal mucosa morphology and carcass traits.

The present study investigated the occurrence of intra-uterine growth retardation (IUGR) in newborn (n=40) and 150-day-old (n=240) pigs of different birthweight ranges (high, HW: 1.8-2.2kg; low, LW: 0.8-1.2kg) from higher-parity commercial sows and its impact on their subsequent development and carcass traits in a Brazilian commercial production system. HW newborn pigs had heavier organs than LW pigs (P<0.01), and all brain:organ weight ratios were higher (P<0.01) in LW compared with HW offspring, providing strong evidence of IUGR in the LW piglets. HW pigs had higher bodyweights and average daily gain (ADG) in all phases of production (P<0.05), but ADG in the finisher phase was similar in both groups. Additionally, LW newborn and 150-day-old pigs showed a lower percentage of muscle fibres and a higher percentage of connective tissue in the semitendinosus muscle, greater fibre number per mm(2) and a lower height of the duodenal mucosa (P<0.05). On the other hand, HW pigs had higher hot carcass weight, meat content in the carcass and yield of ham, shoulder and belly (P<0.01). Hence, lower-birthweight piglets may suffer from IUGR, which impairs their growth performance, muscle accretion, duodenal mucosa morphology and carcass traits.

Consequences of a low litter birth weight phenotype for postnatal lean growth performance and neonatal testicular morphology in the pig.

The consequences of a low litter average birth weight phenotype for postnatal growth performance and carcass quality of all progeny, and testicular development in male offspring, were investigated. Using data from 25 sows with one, and 223 sows with two consecutive farrowing events, individual birth weight (BW) was measured and each litter between 9 and 16 total pigs born was classified as low (LBW), medium (MBW) or high (HBW) birth weight: low and high BW being defined as >1 standard deviation below or above, respectively, the population mean for each litter size. Litter average BW was repeatable within sows. At castration, testicular tissue was collected from 40 male pigs in LBW and HBW litters with individual BW close to their litter average BW and used for histomorphometric analysis. LBW piglets had a lower absolute number of germ cells, Sertoli cells and Leydig cells in their testes and a higher brain : testis weight ratio than HBW piglets. Overall, LBW litters had lower placental weight and higher brain : liver, brain : intestine and brain : Semitendinosus muscle weight ratios than MBW and HBW litters. In the nursery and grow-finish (GF) phase, pigs were kept in pens by BW classification (9 HBW, 17 MBW and 10 LBW pens) with 13 males and 13 females per pen. Average daily gain tended to be lower in LBW than HBW litters in lactation (P = 0.06) and throughout the nursery and GF phases (P < 0.01), resulting in an increasing difference in body weight between LBW, MBW and HBW litters (P < 0.05). Average daily feed intake was lower (P < 0.001) in LBW than HBW litters in the nursery and GF phases. Feed utilization efficiency (feed/gain) was similar for LBW and HBW litters in the nursery, but was lower (P < 0.001) in HBW than LBW litters in the GF phase. By design, slaughter weight was similar between BW classifications; however, LBW litters needed 9 more days to reach the same slaughter weight than HBW litters (P < 0.001). BW classification did not affect carcass composition traits. In conclusion, LBW litters showed benchmarks of intrauterine growth retardation, LBW had a negative impact on testicular development and germ and somatic cell populations, and was associated with decreased postnatal growth during all phases of production; however, no measurable effect on carcass composition traits was established.

Glycol Methacrylate Embedding for Improved Morphological, Morphometrical, and Immunohistochemical Investigations Under Light Microscopy: Testes as a Model

Glycol methacrylate (GMA), a water and ethanol miscible plastic resin, is a medium handy to use for light microscopy embedding that has a number of advantages than paraffin embedding. The GMA improves the histological, morphometrical, and immunohistochemical evaluations, mainly due to the accurate assessment of cytological details. This chapter focuses on our experience in the GMA processing and describes in detail the fixation, embedding, and staining methods that we have been using for testes evaluations.

Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone suppression.

Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.

Available phosphorus levels in diets for barrows at 60 to 95 kg

The effects of available phosphorus levels on performance and carcass composition of barrows with high genetic potential for meat deposition were evaluated. Sixty swine with initial body weight of 59.84 ± 1.64 kg were assigned to a randomized block experimental design, with five diets (0.097; 0.190; 0.280; 0.370 and 0.460% aP), six replicates and two animals per experimental unit. Experimental diets and water were supplied ad libitum until the end of the experimental period, when animals reached 96.64 ± 3.68 kg. Weight gain and feed conversion improved in a quadratic way until the estimate levels 0.35 and 0.33% aP in the diets, respectively. There was no effect of the diets on daily feed intake. Consumption of available phosphorus linearly increased as levels of this mineral increased in the diet. The diets did not change activity of alkaline phosphatase enzym. However, values of serum inorganic phosphorus at 21 days of age and in the end of the experimental period increased in a quadratic way until the estimate level 0.35 and 0.38% in the diet, respectively. Levels of available phosphorus affected in a quadratic way loin depth, daily fat-free lean deposition rate, and the amount of fat-free lean which improved until the estimated level of 0.35; 0.31 and 0.33% of aP in the diet, respectively. In the phase from 60 to 95 kg, the level of 0.33% of aP in the diet, which corresponds to a 9.38 aP/day intake provided better feed conversion and a greater amount of fat-free lean meat in hybrid commercial barrows genetically selected for meat deposition on the carcass.

Revisiting the human seminiferous epithelium cycle

STUDY QUESTION Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? SUMMARY ANSWER By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. WHAT IS ALREADY KNOWN Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. STUDY DESIGN, SIZE, DURATION Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levins, Johnsens and Bergmanns scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. MAIN RESULTS AND THE ROLE OF CHANCE HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. LIMITATIONS, REASONS FOR CAUTION Occasionally, germ cells can be absent in some tubular sections. In this situation, it has to be taken into account the germ cell association proposed in the present study to classify the stages. WIDER IMPLICATIONS OF THE FINDINGS Our findings bring a new focus on the morphology and development of germ cells during the SEC in human. Application of HRLM may be a valuable tool for research studies and clinical andrology helping to understand some testicular diseases and infertility conditions which remain unsolved. STUDY FUNDING/COMPETING INTEREST Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. TRIAL REGISTRATION NUMBER Not applicable.

Influence of three different histological methods on the morphology and morphometrical data in human testis

Coagulant fixatives and paraffin embedding were widely used in the past for histomorphometrical evaluations of the human testis under physiological and pathological conditions. However, new methods are applied nowadays using better combinations of fixatives and plastic resins as embedding media, improving cell and tissue structural preservation. In an attempt to compare old and new data, the present study evaluated histomorphometrical data obtained from human testis after three different histological processing methods: Bouin/paraplast, glutaraldehyde/glycol methacrylate and glutaraldehyde/araldite. The morphometrical parameters were not affected by glutaraldehyde fixation after both resin embedding (methacrylate or araldite). On the other hand, Bouin/paraplast embedding lead to tissue shrinkage, which could give rise to misinterpretations on the measurements performed. Since some germ and somatic cells recognition do not depend upon high resolution techniques, counting of such cell types could be performed even using routine Bouin/paraplast protocols. Thus, the morphometrical analyses relying on cell recognition were not affected by the methods here applied, however, when metric measurements were applied, the obtained results could not be promptly compared. However, if the study requires confident spermatogonial identification for kinetics evaluation, glutaraldehyde/araldite processing is highly recommended.

Effects of birthweight on reproductive system development and onset of puberty in gilts

The aim of the present study was to investigate the effects of birthweight on bodyweight development, development of the genital tract, onset of puberty and their associations with insulin-like growth factor (IGF) 1 and leptin concentrations. Pairs of littermate gilts from 51 litters were selected: one piglet with the highest birthweight (HW; 1.5±0.2kg) and the other with the lowest birthweight (LW; 1.0±0.2kg). Gilt pairs were killed at either fixed ages (80.8±1.2 days; AG; 16 pairs), fixed bodyweight (35.2±1.4kg; WG; 16 pairs) or after first oestrus (EG; 19 pairs). In the AG group, HW gilts were 5.6kg heavier at the time of death than LW gilts. In the WG group, LW gilts were 5.9 days older at the time of death (P<0.05). There were no significant differences in the number or size of total antral follicles or in the follicle population among birthweight classes. Age at puberty was similar between the HW and LW gilts, but bodyweight at time of death was greater for HW gilts (P<0.05). Birthweight did not affect the development of the genital tract, ovulation rate or hormone plasma concentrations. These results suggest that birthweight does not affect the development of the genital tract before puberty and puberty onset.

Vascularization of broad ligament of uterus and its relationship with fetal and placental development in gilts.

The objective of this study was to evaluate the influence of vascular architecture of broad ligament of the uterus on fetal and placental development in gilts. Fifteen gilts DB-90 (DanBred) were divided into three groups according to gestational age at slaughter (50, 80, and 106 days). After slaughter, fetuses and placentas were collected, weighed, and measured. The uterine arterial system was detached by latex repletion for quantification of the number and diameter of the terminal vessels in different regions of the uterine horns (apex, middle region, and base). Fetal and placental measurements were statistically analyzed and correlated with the number and diameter of arteries in each uterine segment. No correlation was observed (P > 0.10) between the number and diameter of arteries destined to the uterus with the number or weight of fetuses or placental weight in any gestational group. It was observed (P < 0.05) that more vessels destined to the medium region of the uterine horns, independent of the gestational age or uterus side. At the 80th day of gestation, fetuses located at the base of the uterus have (P < 0.05) smaller cephalic and thoracic perimeters. It was concluded that there were differences in vascularization of broad ligament that irrigates the different uterine segments, but this was not sufficient to influence the development of fetuses in gilts. The middle region of the uterine horns was the segment with a greater number of vessels, regardless of gestational age.

Spermatogenesis recovery in protein-restricted rats subjected to a normal protein diet after weaning

This study investigated the pre- and postnatal effects of protein restriction (8% vs 20% crude protein) on different parameters of spermatogenesis in adult rat offspring. Body and testis weights as well as the seminiferous tubular diameter were reduced in those animals that received the protein-restricted diet after weaning, although these parameters recovered when a 20% protein diet was offered subsequently. The numbers of spermatogonia, spermatocytes, spermatids and Leydig cells were reduced in undernourished animals, whilst the Sertoli cell number did not change. Prenatal programming effect was observed only in the spermatogonial or proliferative phase of spermatogenesis. However, the intake of the normal protein diet after weaning brought many of the testicular parameters evaluated back to normal in 70-day-old rats. A significant reduction of the meiotic index, Sertoli cell supporting capacity and spermatogenic efficiency was observed in animals subjected to protein undernutrition throughout their lives. The data presented show that protein restriction impairs the normal development of the testis in different ways, depending on the period during which the restriction was imposed, and the negative effects on spermatogenesis are more severe when undernutrition occurs from conception to adulthood; however, the return to a normal protein diet after weaning recovers the spermatogenic process.