Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen.

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs = 47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n = 6; fresh epididymal: n = 6; frozen-thawed ejaculated: n = 6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen-thawed samples (Exp. I and II; p < 0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI-motility, p = 0.01; SDI-acrosome integrity, p = 0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.

A dry powder formulation from silk fibroin microspheres as a topical auto-gelling device.

With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.

Formulation of microspheres containing Crataegus monogyna Jacq. extract with free radical scavenging activity.

Extracts of Crataegus monogyna Jacq. (hawthorn) show an interesting free radical scavenging (FRS) effect, related to their flavonoids content. Unfortunately, their oral administration is affected by their low bioavailability. The aim of this work is to obtain a multiparticulate drug delivery system for hawthorn extracts for oral administration. The extracts from flowering tops (FL) or fruits (FR) of hawthorn were obtained with maceration, using ethanol as an extraction solvent, and their antioxidant activity was evaluated. FL extract showed the highest FRS activity (EC50 3.72 ± 1.21 µg/ml), so it was selected to prepare microparticulate systems by a spray-drying technique, which were characterized by granulometric analysis, scanning electron microscopy-energy dispersive X-ray spectroscopy, confocal fluorescence microscopy and hyperoside content. Antioxidant activity was evaluated before and after gastrointestinal transit in vitro simulation. Results indicate that the microparticulate systems maintained the antioxidant activity of hawthorn also after gastrointestinal transit in vitro simulation, exhibiting properties suitable for oral administration.

Bioencapsulation of Oocytes and Granulosa Cells.

A protocol for the encapsulation in sodium alginate of granulosa cells in primary culture and coculture of oocyte-cumulus complexes is reported. Sodium alginate forms strong gels when jellified with barium ions, allowing the self-organization of cells into a 3D structure. This method of encapsulation is simple and cheap, allowing the culture of cells in a three-dimensional fashion.

Apgar Score for Newborn Dog Viability Assessment: Differences between English and French Bulldogs Born via Cesarean Section.

Even if largely used in canine neonatology, some questions about the Apgar Score (AS) arose. Notably, considering the breed-oriented modifications of the score are needed, slight changes of the score were reported for English (EBs) and French bulldogs (FBs). The present study aimed to evaluate the possible differences between neonatal viability of the two breeds assessed by AS in puppies born via cesarean section. The results obtained from 99 puppies born alive and without malformations (EB = 47, FB = 52) showed significant (p < 0.001) differences in the median AS (seven in EB vs. eight in FB), with Grimace (p < 0.05 for sub-score 0 and p < 0.001 for sub-score 2) and Attitude (p < 0.05) being differently sub-scored between the two breeds. In the 89 alive puppies at 7 days of age, the same difference in the median AS was observed (p < 0.001), and only Grimace was differently sub-scored between the two breeds (p < 0.05 for sub-score 0 and p < 0.01 for sub-score 2). These results suggest that low Grimace could be an intrinsic characteristic of EB newborns, but it could negatively affect the neonatal adaptation process of EBs, providing an indication for focused neonatal assistance. Neonatal mortality at 7 days of age was higher in EBs than in FBs (17 vs. 3.8%, respectively), which deserves further investigations. The study provides further evidence that breed-oriented ASs are needed for a better evaluation and assistance of purebred puppies at birth.

Coat, Claw and Dewclaw 17-ß-Estradiol and Testosterone Concentrations in Male and Female Postpubertal Cats: Preliminary Results.

In the recent past, tissue materials such as hair/coat and nails/claws have proved to be useful for the study of long-term hormonal changes in humans and animals and shown to be advantageous in terms of being collectable without invasiveness, with a benefit in terms of animal welfare. However, studies using these tissue materials in cats are scarce, especially on sexual hormone measurement. In this study, the concentrations of 17-ß-estradiol (E2) and testosterone (T) were assessed in 20 male and 18 female domestic postpubertal cats at the time of neutering/spaying during the breeding season. Hormones were measured in coat shaved from the forearm (ACOAT) and from the surgical area (SCOAT); claws were collected from the forearms (CLAWS) and the dewclaws (DCLAWS). Although all these tissue materials were shown to be useful for E2 and T long-term measurement, only T concentrations were higher (p < 0.001) in males from both ACOAT and SCOAT samples when compared to females and therefore useful for distinguishing between the two sexes. Within each sex, E2 and T concentrations can be assessed on coat, but also on the dewclaws, providing an alternative, practical, matrix for sexual steroid measurement in postpubertal cats during the breeding season.

Chemical Characterization and In Vitro Gas Production Kinetics of Alternative Feed Resources for Small Ruminants in the Maltese Islands.

The ever-increasing human population, the problem associated with climate change and recent crises-COVID-19 disease and trade conflicts-all impacted on the availability and cost of animal feed raw materials. This is clearly visible in realities which heavily rely on importation such as islands and small states, where producers involved in the agricultural sector were strongly affected by the sharp increase in prices. To deal with these global issues, alternative resources are perceived to replace conventional ingredients. This work aimed at assessing the nutritive value of different resources (sheep feed, mature carob, Maltese bread, wild asparagus, prickly lettuce, and loquat) for small ruminants present in the Maltese Islands, analyzing their chemical composition, gas production kinetics and antioxidant properties. In general, the variation in chemical composition resulted in different rumen fermentation kinetics (p < 0.007). The ratio between GP-24 h and GP-48 h was higher in Maltese bread than other substrates; loquat, prickly lettuce and wild asparagus showed lower fermentation kinetics in accordance with their high NDF and ADF contents. The antioxidant activity may be partially related to the polyphenolic content that was higher in wild asparagus, prickly lettuce and loquat. All feed characteristic confirmed their potential to be included as ingredients in ruminant diets and as a source of fiber.

Peripubertal Testosterone, 17ß-Estradiol and Progesterone Concentrations in Hair and Nails in Dobermann Dogs.

Studies about puberty in dogs are few, probably because many factors are involved in the delicate process of puberty onset, leading to difficulties in the proper enrollment of subjects. Moreover, the use of blood for monitoring hormonal changes can be problematic, and not feasible for long-term studies. Hair and nails proved to be suitable matrices for the retrospective evaluation of hormones' long-term accumulation. This study was performed using hair and nails for the evaluation of testosterone (T), 17ß-estradiol (E2) and progesterone (P4) concentrations to assess possible sexual steroid changes during the peripubertal period in dogs. The results, obtained on five males and five females, showed a significant increase in T in hair and nails of males immediately before puberty. In females, a significant increase in E2 at puberty and a marked increase in P4 after puberty was found in both biological specimens. Sex-related differences were detected only for T hair concentrations, but when the sex and sampling time were considered together, hair and nails T and nails P4 concentrations allowed us to discern between male and female dogs at specific sampling times. The results from this study showed that hair and nails are useful biological specimens for the retrospective evaluation of changes in T, E2 and P4 concentrations in peripubertal dogs.

Partial Replacement of Synthetic Vitamin E by Polyphenols in Post-Weaning Piglets.

Vitamin E is an essential nutrient usually recommended in post-weaning piglets, when a decline in the serum vitamin E concentration is observed. Selected polyphenols have the potential to partially replace vitamin E in animal feed. The aim of this study was to investigate the effect of the dietary inclusion of some commercial polyphenol products (PPs) on the growth performance, antioxidant status and immunity of post-weaning piglets. A total of 300 piglets (BW 7.18 kg ± 1.18) were randomly assigned to six dietary groups: CON- (40 mg/kg vitamin E); CON+(175.8 mg/kg vitamin E); and PP1, PP2, PP3 and PP4, in which 50% vitamin E of CON+ was replaced with PP with equivalent vitamin E activity. The PP1 group exhibited lower performance (p < 0.05) than the other dietary groups, but a similar performance to that commonly registered in pig farms. Dietary polyphenols did not influence the IgG concentration or the IL-6, IL-10, IFN-γ and TNF-α cytokine concentrations. A lower IL-8 level was found in the PP4 group than in the other groups. The diets that affected the vitamin A content showed the highest value (p < 0.05) in the PP1 group, and a trend was noted for vitamin E with a higher content in PP4 and CON+. The polyphenols-enriched diets, especially the PP3 diet, maintained an antioxidant capacity (whole blood KRL) similar to the CON+ diet. In conclusion, the replacement of vitamin E with all PPs enables partial vitamin E substitution in post-weaning piglets.

Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet.

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

Artificial neural networks for the definition of kinetic subpopulations in electroejaculated and epididymal spermatozoa in the domestic cat.

This study was designed for the identification of different sperm kinetic subpopulations in feline semen using artificial neural networks (ANNs) and for the evaluation of the effect of ejaculation on motility patterns of these subpopulations. Seven tomcats presented for routine orchiectomy were electroejaculated, and after 5 days, orchiectomized and epididymal tail sperms were collected. Sperm motility characteristics were evaluated using a computer-assisted sperm analyzer that provided individual kinetic characteristics of each spermatozoon. A total of 23 400 spermatozoa for electroejaculated and 9200 for epididymal tail samples were evaluated using a multivariate approach, comprising principal component analysis and ANN classification. The multivariate approach allowed the identification and characterization of three different and well-defined sperm subpopulations. There were significant differences before (epididymal tail spermatozoa) and after (electroejaculated sperm) ejaculation in sperm kinetic subpopulation characteristics. In both epididymal and ejaculated samples, the majority of subpopulation was characterized by high velocity and progressiveness; however, the electroejaculated samples showed significantly higher values, suggesting that the microenvironment of the epididymal tail could affect the sperm motility or, alternatively, seminal plasma could increase the kinetic characteristics of the spermatozoa, indicating that only after ejaculation, the spermatozoa express their motility potential. Nevertheless, further studies are required to clarify the functional significance of each kinetic subpopulation.

Refining the APGAR Score Cutoff Values and Viability Classes According to Breed Body Size in Newborn Dogs.

The Apgar score (AS) represents a key tool for neonate assessment, but the possible breed effect on AS in newborn puppies has never been investigated. Therefore, data from 234 dog litters born by caesarean section, grouped according to breed body size (BBS) (small, medium, large), were evaluated. Live-birth puppies were assessed through AS within 5 min of delivery, and classified in viability classes: 0−3 severely distressed, 4−6 moderately distressed, 7−10 not distressed. Statistical analysis evaluated possible differences of AS and viability class according to BBS, and between BBS and puppies' mortality. Results showed no differences in the distribution of mortalities among BBSs. However, an effect of BBS on the AS was found, with small-sized puppies being the most represented in the severely distressed class, but having the best survival chance compared to large-sized newborns. Through receiver-operating-characteristics analysis, the AS new cutoff values for survival and for death <24 h and 24 h−7 days of age were identified, and the viability classes were redefined, with a narrower class of moderately distressed puppy specific for each BBS. In conclusion, the refining of the AS in dog species is imperative, with cutoff values and viability classifications that must be adapted to the BBS.

Hair and Claw Dehydroepiandrosterone Concentrations in Newborn Puppies Spontaneously Dead within 30 Days of Age.

The latest intrauterine fetal developmental stage and the neonatal period represent the most challenging phases for mammalian offspring. Toward the term of pregnancy, during parturition, and after birth, the hypothalamic−pituitary−adrenal axis (HPA) is a key system regulating several physiologic processes, through the production of cortisol and dehydroepiandrosterone (DHEA). This study was aimed to assess DHEA concentrations in hair and claws of 126 spontaneously dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). The possible influence of newborn sex, breed body size, and timing of death on DHEA concentrations in both matrices was evaluated. Claw DHEA concentrations were higher in the PRE-P group when compared to STILL-P and NEON-P puppies (p < 0.05), whilst no significant differences were found in hair for all the studied factors. The results confirm the hypothesis that higher amounts of DHEA are produced during the intrauterine life in dogs, also in puppies that will die soon after birth.

Investigating the Behavior and Personality Structure of the Aldabra Tortoise during Human Interactions and Training Events.

Human-animal interaction (HAI) can be valuable for captive animals, and many zoo-housed species benefit from interactions with their keepers. There is also an increasing body of evidence that some animal species possess personalities that are temporally consistent. However, the majority of zoo research, particularly on personality and HAI, traditionally has focused on mammals, and there are comparatively fewer studies on reptiles. Research was undertaken at the Faunistic Park Le Cornelle, Italy, to investigate the effects of approach tests and food interaction events on 5 male and 5 female Aldabra tortoise (Aldrabrachelys gigantea) behavior. During human-tortoise interactions, continuous focal sampling of behavior took place. The behavioral differences between individual tortoises, interaction type (approach or training) and person involved in the interaction (keeper, vet, or observer) were analysed using general linear mixed-effects models. The personality components of the tortoises were also quantified using principal component analysis. Overall, the study revealed that individuals acted significantly differently to one another, and that while females initially appeared to display a greater number of shy behaviors, this was not consistent across the group. PCA revealed two personality dimensions in the tortoises: boldness and avoidance. Overall, the study revealed that the tortoises possessed personalities that were independent of sex or scenario. The study also revealed that many tortoises chose to interact with their keepers during training and approach tests: this suggests that human-tortoise interactions may have some enrichment value. Future research could quantify tortoise personality in other scenarios such as in social interactions with other tortoises.

Comparison of Female Verzaschese and Camosciata delle Alpi Goats' Hematological Parameters in The Context of Adaptation to Local Environmental Conditions in Semi-Extensive Systems in Italy.

Local livestock breeds are pivotal to ensure sustainable agriculture and represent a real genetic resource in the context of genetic variability reduction. This study aimed at broadening the knowledge about hematological values in female Verzaschese and Camosciata delle Alpi goats (an Italian local and a cosmopolitan goat breed, respectively) and investigating factors affecting them (i.e., breed, age, season). Blood samples were collected monthly from 34 Verzaschese and 37 Camosciata delle Alpi female goats kept under the same semi-extensive farming system for a whole year. The main hematological parameters were evaluated, and descriptive as well as inferential statistical analyses were performed. Reference intervals for complete blood cell count were established and several inter-breed differences were found. In particular, most of the red blood cell parameters were higher in Verzaschese than Camosciata delle Alpi (p < 0.05) suggesting a greater gastrointestinal parasites' resilience of the local breed. The age effect (p < 0.05) was consistent with the literature while the season effect (p < 0.05) could be explained by the melatonin immunostimulant action and gastrointestinal parasitism influence. Overall, differences in blood values could be attributed to physiological changes and adaptive strategies developed through centuries highlighting the remarkable rusticity and adaptation to the environment and farming system of the local breed.

How Stressful Is Maternity? Study about Cortisol and Dehydroepiandrosterone-Sulfate Coat and Claws Concentrations in Female Dogs from Mating to 60 Days Post-Partum.

In dogs, the phase from mating to the end of weaning lasts about 120 days and encompasses many aspects that, interacting, contribute to increase the allostatic load. The coat and claws, useful for long-term change assessments, have the advantage of being collectable without invasiveness. In the present study, the Cortisol (C) and Dehydroepiandrosterone-sulfate (DHEA-S) concentration monthly changes in the coat and claws were studied in female dogs from mating to the end of weaning to assess Hypothalamic-Pituitary-Adrenal (HPA) axis activation during pregnancy and the post-partum period. The results from 15 Dobermann Pinscher female dogs showed a trend of increase of the coat C from mating to 60 days post-partum, with significant changes between mating and parturition-60 days post-partum (p < 0.01) and between the 30-day pregnancy diagnosis (PD) and 30-60 days post-partum (p < 0.05). The claws C trend showed significant increases between mating and 30-60 days post-partum (p < 0.05) and between the PD and 60 days post-partum (p < 0.01). DHEA-S in both matrices showed non-significant changes. The results suggest that maternity could play a pivotal role in the HPA axis activation, with a subsequent chronic secretion of C determining an increase in the allostatic load in the mothers. Neither maternal parity nor litter size played a significant role in the accumulation of C and DHEA-S in both matrices.

Clinical Trial on the Usefulness of On-Site Evaluation of Canine Fetal Fluids by Reagent Test Strip in Puppies at Elective Caesarean Section.

The reagent urinary test strips (TS) marketed for urines represent the first-line diagnostic tool in many instances. Therefore, the aim of the study was to assess the usefulness of TS for the on-field evaluation of fetal fluids' composition in newborn dogs at elective caesarean section. Of a total of 137 puppies born at term, 127 survived and 10 did not survive. One hundred and thirteen amniotic and 107 allantoic samples from surviving newborns were collected, and 8 amniotic and allantoic fluids were collected from the non-surviving newborns and assessed by strips. Significantly lower amounts of amniotic glucose and higher amounts of amniotic and allantoic nitrites, amniotic protein, allantoic urobilinogen, and amniotic bilirubin concentrations were found in non-surviving when compared to surviving newborns. In the surviving ones, higher specific gravity and bilirubin concentrations, and lower pH, were found in allantoic than in amniotic fluids. Higher amniotic and allantoic glucose concentrations, higher amniotic and allantoic pH, and lower amniotic and allantoic protein concentrations were found in medium/large- than in small-sized puppies. The TS allowed the quick evaluation of fetal fluids in puppies at birth. The differences between surviving and non-surviving puppies seem to suggest that the on-site analysis of fetal fluids' composition by TS could represent a first-line diagnostic tool in the field of canine neonatology, allowing the quick recognition of puppies needing assistance as a complementary tool for clinical evaluation.

Effect of Delivery by Emergency or Elective Cesarean Section on Nitric Oxide Metabolites and Cortisol Amniotic Concentrations in at Term Normal Newborn Dogs: Preliminary Results.

The neonatal response to stress was reported to be related to the type of delivery, that is elective or emergency cesarean sections (ELCS and EMCS, respectively). Nitric oxide (NO) is also reported to be related to uterine inertia, and high levels of NO metabolites (NOs) are associated with physical and emotional stress. The study aimed to assess the concentrations of cortisol (C) and NOs in the amniotic fluid of puppies delivered by ELCS or EMCS. In total, 32 puppies were delivered by ELCS and 22 by EMCS. ANCOVA showed an effect of the ELCS vs. EMCS on both amniotic NOs (p < 0.001) and C (p < 0.001) concentrations. Lower amniotic C concentrations were found at increasing Apgar score (p < 0.001). Higher amniotic NOs concentrations were associated to increasing mother's parity (p < 0.001), puppies' birthweight (p < 0.001), and time of labor within the EMCS group (p < 0.05). A positive correlation between birthweight and amniotic NOs concentrations was also found (p < 0.05) in the EMCS group. Due to the possible concurrence of several compartments (maternal, maybe placental, and fetal) to the final amniotic fluid composition, the definition of the role played by the three compartments in the higher C and NOs concentrations found in amniotic fluids collected from puppies delivered by EMCS than ELCS needs further clarification.

Effect of Goji Berry (Lycium barbarum) Supplementation on Reproductive Performance of Rabbit Does.

Goji berry shows a wide range of beneficial properties in human health, but only a few studies evaluated its effects in livestock animals. The objective of this research was to assess the effects of goji berry supplementation on the hormonal profile, productive, and reproductive performance of does. Two months before artificial insemination, 105 nulliparous does were randomly divided into three groups (n = 35) based on the dietary treatment: commercial diet (C), or a diet supplemented with either 1% (G1), or 3% (G3) of goji berry, respectively. The results showed that receptivity was higher in G1 than in the C group (p < 0.05). Trends toward significance for differences between the G1 and G3 groups in marginal means of LH concentrations (p = 0.059), and between G1 and C in LH AUC values (p = 0.078), were evidenced. Estrogen concentrations showed a more fluctuating trend but a significant interaction effect (p < 0.001). The G1 group showed higher litter weight than C at birth (p = 0.008) and weaning (p < 0.001), as well as higher litter size at weaning (p = 0.020). The G1 group also exhibited the highest mean milk production (p < 0.01). In conclusion, goji berry influenced reproductive and productive performance, probably via modulating hormonal patterns and milk production in rabbits. However, further studies are needed to validate these preliminary results.

Apgar score or birthweight in Chihuahua dogs born by elective Caesarean section: which is the best predictor of the survival at 24 h after birth?

BACKGROUND: In the dog, the correct management of parturition and the prompt neonatal evaluation and assistance can reduce the perinatal mortality rates that are particularly high in toy breeds. Newborn evaluation and factors addressing prognosis are pivotal to guarantee the correct neonatal assistance. Assessment of the Apgar score with viability classification and birthweight are recognized as predictors for neonatal survival in dogs, but breed-specific data are needed for a more feasible application in the dog species, in which wide differences among breeds are known. The present study aimed therefore to: (a) assess the role of Apgar score and birthweight as predictors for the survival of Chihuahua newborn puppies in the first 24 h of life; (b) to assess a cut-off of the Apgar score and birthweight values that can predict the survival of Chihuahua newborn puppies in the first 24 h after birth; (c) to assess the possible effect played by maternal parity, newborn gender and litter-size on Apgar score in Chihuahua newborn puppies, in order to provide breed-specific data for a better neonatal assistance. RESULTS: Data obtained from 176 normal developed Chihuahua puppies born by elective Caesarean section, showed that 62%, 28% and 10% of puppies were classified in the Apgar score classes 7-10, 4-6 and 0-3, respectively, with survival at 24 h after birth of 97%, 96%, 39%, in the three Apgar classes of viability, respectively. Apgar score was a better predictor for survival at 24 h after birth than birthweight (AUC 0.93, P < 0.0001; AUC 0.69, P < 0.01, respectively). Litter-size of 7 puppies/litter plays a negative effect on Apgar score. Apgar score is a better predictor of survival at 24 h than birthweight, and the best cut-off of Apgar score for survival at 24 h after birth is 4, with 96% sensitivity and 77% specificity. CONCLUSIONS: The different proportion of "normal viable" and "less viable" neonates in comparison to other studies highlights that Chihuahua puppies born by elective Caesarean section should be carefully evaluated at birth to provide correct assistance.