Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

The relationship between acrosome reaction and polyunsaturated fatty acid composition in boar sperm

This study investigated the relationship between acrosome reactions and fatty acid composition with respect to fertility in boar sperm. The acrosome reaction was induced more than 85% by 60 mM methyl-beta-cyclodextrin (MBCD), and plasma membrane integrity was significantly reduced dependent on the MBCD level in boar sperm (p < .05). The acrosome-reacted sperm exhibited significantly higher saturated fatty acids (SFAs) and lower polyunsaturated fatty acids (PUFAs) composition compared to the non-acrosome reaction group (p < .0001). In addition, the PUFAs, C22:5n-6 (docosapentaenoic acid [DPA]; p < .01) and C22:6n-3 (docosahexaenoic acid [DHA]; p < .0001) were significantly decreased, and cleavage and blastocyst formation of oocytes were significantly (p < .0001) decreased in acrosome-reacted sperm relative to non-acrosome-reacted sperm. Moreover, acrosome reaction was positively correlated with SFAs, whereas negatively correlated with PUFAs, of the PUFAs, the DPA (p = .0005) and DHA (p = <.0001) were negatively correlated with the acrosome reaction. Therefore, these results suggest that the PUFAs composition of sperm is closely involved in acrosome reaction in pigs.     

Bovine xenograft application for treatment of a metatarsal nonunion fracture in an alpaca (Vicugna pacos)

CASE HISTORY: A 15-year-old female huacaya alpaca (Vicugna pacos) was referred because of a non-weight-bearing lameness (4/4) in the left pelvic limb caused by a grade three open metatarsal fracture. The referring veterinarian treated the fracture with conservative management using bandages, but it progressively evolved to a non-union.

CLINICAL FINDINGS AND DIAGNOSIS: Clinical examination revealed external wounds on the medial and lateral surfaces of the metatarsus. Radiographs confirmed an open, nonarticular, displaced, diaphyseal fracture of the left metatarsus.

TREATMENT AND OUTCOME: Cancellous bone was sourced from bovine proximal and distal femur epiphyses, followed by a thermal shock procedure to achieve decellularisation, to produce a xenograft. Open reduction and internal fixation of the fracture using locking plates was performed. Alignment of the fracture fragments was corrected and the xenograft was placed at the debrided fracture site to stimulate and harness osteogenesis in situ. Clinical and radiographic follow-up was performed up to 40 weeks postoperatively. Clinical evaluations revealed that the alpaca gradually increased weight bearing following bandage removal 10 days after surgery. Serial radiographs showed correct alignment of the left metatarsus, progressive bone modelling and, complete bone union at 12 weeks. Ten months postoperatively the alpaca showed no signs of lameness and resumed normal activity.

CLINICAL RELEVANCE: For management of a metatarsal non-union, a combination of bovine xenograft application and angular stable internal fixation progressed toward an excellent long-term recovery.     

Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos)

The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G‐coupled receptors named receptor 1 (OX₁) and receptor 2 (OX₂) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX₂ by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX₂‐IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX₂. Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.     

Pulmonary alveolar microlithiasis in an alpaca (Vicugna pacos)

Pulmonary alveolar microlithiasis (PAM) is a rare, familial disease of unknown aetiology characterised by intra‐alveolar formation and accumulation of microliths. Multiple formalin‐fixed tissues were submitted from a 5‐month‐old female alpaca that died suddenly without significant clinical signs. No gross abnormalities were observed on postmortem examination. Histological findings included PAM and severe centrilobular hepatic necrosis. Although the hepatic lesion was the likely cause of death, PAM was an incidental finding that has not been reported previously in alpacas. An overview of PAM, including pathogenesis and histopathological characteristics, are discussed in relation to the concurrent hepatic disease in the present case.     

Diaphragmatic hernia in a two-year-old alpaca (Vicugna pacos)

A 2-yr-old male intact alpaca (Vicugna pacos) was admitted for a 4-day history of anorexia and colic. Five months prior, the alpaca had undergone surgical removal of a duodenal trichophytobezoar and had recovered uneventfully. The alpaca died under anesthesia, and diaphragmatic herniation of the third gastric compartment (C3) was diagnosed at necropsy. A defect was identified in the left dorsal hemidiaphragm accompanied by herniation of 80% of C3 and the aboral portion of the second gastric compartment into the pericardial sac. The smooth margins and dorsal location of the diaphragmatic defect suggested a congenital origin. Diaphragmatic herniation is uncommon in camelids, and only one other case has been reported. Due to the dorsal location of the diaphragmatic defect in this animal, positioning during the previous surgery may have initiated a partial entrapment of gastric compartments, leading to a more complete incarceration between when the animal was discharged and presented again.     

Histological characterization of umbilical cord in alpaca (Vicugna pacos)

The histomorphometric features of umbilical cord constituents in seven foetuses of alpaca (Vicugna pacos) from Cerro de Pasco, Department, Peru, were determined. Sections of 2–5 cm of umbilical cord were fixed in 10% neutral buffered formalin and processed for light microscopy. Standard histological slides stained with haematoxylin and eosin, Masson's trichrome and Van Gieson's trichrome were obtained. Histologically, common features of umbilical artery and vein were observed as well as mucous connective tissue, some cell features that compound this tissue constituted by cells presented features of myofibroblasts. Among most important findings that were observed, the lumen of umbilical vein was obliterated into star‐shaped form with the thinner umbilical artery wall; the smooth muscles and fibroblast were comparatively more in number in umbilical artery than that of umbilical vein, and the tunica media was larger in dimension than the tunica adventitia in umbilical vein. Conclusively, this histological study features an observation of the umbilical cord of alpaca foetuses and shows the similarity between them and those of other mammal species, including dromedaries and South American camelids.     

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

Sperm competition and the evolution of sperm form and function in mammals

Sperm competition is a powerful selective force that has influenced many reproductive traits in males and females although additional evolutionary explanations may help to understand the diversity of mammalian reproduction. Sperm morphology varies considerably in mammals with extreme examples in several rodent lineages in which a wide range of sizes and complex head morphologies have been identified. Mammalian spermatozoa also differ in function, with swimming velocity and trajectory showing much divergence. Underlying processes mediating function have received little attention so far, but differences in timing and proportion of sperm undergoing capacitation or acrosomal exocytosis may be related to variation in signalling processes. Furthermore, energy required for sperm functions (such as motion, signalling and overall maintenance of cell integrity) can be produced and consumed, following different patterns among species and this could be the result of several selective forces. A more thorough understanding of the diversity in structure and function of sperm cells, and underlying selective forces, may help us develop better methods to assess them taking into account particulars and generalities of sperm form and performance. Such tests could then become more reliable in estimations of the impact of cryopreservation or effect of changes in the environment and their relevance for fertility.     

Bilateral iliac arterial thrombosis in an alpaca (Vicugna pacos) cria

A 3-month-old male alpaca was presented for acute onset of non-weight-bearing left hind limb lameness. Antemortem diagnostics revealed a mass to the right of the urinary bladder and no blood flow in the left femoral artery. Necropsy revealed bilateral iliac arterial thrombi and an abscess near the urinary bladder.     

Treatment of a periradicular lesion in an alpaca (Vicugna pacos)

This case report describes the diagnosis and surgical treatment of a cyst-like periradicular lesion in an alpaca.     

Prevalence of Candidatus Mycoplasma haemolamae,bovine viral diarrhoea virus,and gastrointestinal parasitism in a sample of adult New Zealand alpaca (Vicugna pacos)

AIM: To determine the prevalence of infection with Candidatus Mycoplasma haemolamae (Mhl), antibodies to bovine viral diarrhoea virus (BVDV), and BVDV antigen, and the prevalence of animals with elevated faecal nematode egg counts (FEC) in a sample of adult New Zealand alpaca (Vicugna pacos).

METHODS: Blood samples were obtained from 175 alpaca, collected from 15 farms around New Zealand, and from 31 samples sent to a diagnostic laboratory for routine haematology. Blood smears (n=170) were examined microscopically for the presence of haemoplasma, and DNA was extracted from whole blood (n=206) for real-time PCR testing for Mhl. Packed cell volume (PCV) was determined for 193 samples. Serum samples (n=195) were tested for BVDV antibody using ELISA, and for BVDV antigen using a real-time PCR assay. Faecal samples were collected from 143 animals; FEC were measured, and samples pooled for larval culture.

RESULTS: No haemoplasma organisms were present on blood smear examination. Of the 206 blood samples, two (from the same farm) were positive for Mhl by real-time PCR testing, giving a prevalence of infection with Mhl of 0.97%. Of the 195 serum samples tested, four (2.1%) were positive for antibodies to BVDV; animals with BVDV antibodies were from 3/15 (20%) farms, none of which farmed cattle. None of the serum samples were positive by PCR for BVDV antigen. The median FEC was 50?epg (min 0, max 4,700), with 55/143 (38.5%) samples having 0?epg, and 33/143 (23.1%) having ≥250?epg. Haemonchus spp. were the most common nematodes present in faecal larval cultures from the North Island. Log₁₀ FEC was negatively associated with PCV (p=0.02), and was higher in males than females (p<0.001), and in animals that were positive compared with negative for Mhl (p=0.022).

CONCLUSIONS AND CLINICAL RELEVANCE: The number of alpaca infected with Mhl was low, as was the seroprevalence of BVDV. Gastrointestinal parasitism was, however, a common finding in this sample of New Zealand alpaca.     

精子活率与精子完整率和质膜完整率的相关性分析

采用考马斯亮蓝（R250）染色法和低渗膨胀试验（HOST试验）检测精子顶体反应和质膜完整性,以检测和评价这2种方法在猪精液质量检测中的实际应用价值。结果表明,冷冻复苏后,精子活率为37.97%,精子平均速度为55.72μm/s,顶体完整率和质膜完整率分别为51.01%和39.95%。经相关性分析得出,猪精子顶体反应率和质膜完整性与精子活率相关系数为0.904 0和0.817 6,呈显著相关（P〈0.01）,而精子的平均游动速度与顶体反应率和质膜完整性相关系数为-0.056 2和-0.151 8,无明显相关性（P〉0.05）,精子活率与精子平均游动速度间也无明显相关性（P〉0.05）。     

Ocular and olfactory forebrain abnormalities within a neonatal alpaca (Vicugna pacos)

A 5-day-old Huacaya alpaca cria (Vicugna pacos) was euthanized due to deteriorating health. At birth, the cria had ophthalmologic abnormalities, but had appropriate mentation. At 2 days of age, the cria gradually stopped suckling and began to circle. At 5 days old, the owner elected euthanasia due to declining clinical condition. Grossly, the right iris had a scalloped pupillary margin, and the right olfactory bulb was malformed. Histopathology revealed persistent hyperplastic primary vitreous bilaterally and iridal abnormalities, as well as aplasia of the olfactory ventricle, olfactory tract, and olfactory foramen on the right side.     

Surgical treatment of scapulohumeral subluxation in an alpaca (Vicugna pacos) using osteotomy of the acromion,open reduction and extracapsular tension sutures

CASE HISTORY AND CLINICAL FINDINGS: A 3.3-year-old male alpaca, weighing 60?kg was referred for investigation of a severe left forelimb lameness of 4 weeks duration. A scapulohumeral subluxation had been diagnosed radiographically by the referring veterinarian.

CLINICAL FINDINGS AND DIAGNOSIS: Based on clinical, ultrasonographic and radiographic findings the diagnosis of cranio-lateral subluxation of the left humeral head was confirmed. In addition, a full thickness lesion (approximately 1?×?1?cm) of the articular cartilage on the caudomedial aspect of the humeral head was diagnosed by arthroscopy.

TREATMENT AND OUTCOME: Treatment included open reduction with internal fixation. Severe muscle contraction and local tissue fibrosis around the scapulohumeral joint (SHJ) required osteotomy of the acromion 3?cm proximal to the distal acromial edge, to allow adequate access. Internal stabilisation was achieved by placing tension band sutures between one cortical screw in the scapular neck and two cortical screws, with washers, craniolaterally on the greater tubercle of the humerus. Post-surgery, a carpal flexion sling was applied with the carpus maintained in 70° flexion for 4 weeks to avoid postoperative weight-bearing. An exercise programme was started 8 days after surgery and continued for 12 weeks. The alpaca had an uneventful postsurgical recovery and showed no lameness after 8 weeks. The long-term outcome was excellent; 21 months after surgery the alpaca was sound and the range of movement of the left SHJ was equal to the right SHJ.

CLINICAL RELEVANCE: Even in this chronic case of subluxation of the SHJ of 4 weeks duration, surgical treatment using osteotomy of the acromion, open reduction and internal fixation with extracapsular scapulohumeral tension sutures resulted in an excellent long-term outcome in this alpaca, despite the presence of a cartilage lesion.