Viability duration of pacu (Piaractus mesopotamicus) milt stored under refrigeration

Duração da viabilidade do sêmen de pacu (Piaractus mesopotamicus) armazenado sob refrigeração

Abstract

Cooling milt conserves viable spermatozoa to extend the period available for artificial fertilization and avoids the robust protocols and high costs associated with cryopreservation. Yet, the sperm quality curves of fresh and refrigerated milt have not yet been compared for pacu (Piaractus mesopotamicus), which is often used as a biological model. This study aimed to analyze the milt quality of male P. mesopotamicus across 24 h of refrigeration. Six adult males were induced with carp pituitary extract. Sperm movement, membrane integrity, and morphology was compared between extruded milt samples stored for 24 h under either ambient temperature or under refrigeration at 12.63 °C. Sperm motility differed significantly over time. After 24 h of storage, motility values were higher in refrigerated spermatozoa than in those kept at ambient temperature. Sperm cell survival rates did not differ 4–8 h post collection. After 16 h, refrigerated cells showed superior membrane integrity (82.05 ± 4.23%) compared to those stored at ambient temperature (66.98 ± 6.45%), maintaining this pattern up to 24 h. In terms of sperm morphology rate, milt from both treatment groups was still viable for use 8 h after collection. However, after 16 h of storage, both groups exhibited a large reduction in normality rates, and at 24 h, all milt were unfeasible. In conclusion, P. mesopotamicus milt can be stored up to 8 h after collection when refrigerated at 12.63 °C, without the use of extenders and/or cryoprotectants, maintaining enough quality for egg fertilization.

Keywords:
chilled milt; computer-assisted sperm analysis; fish reproduction; rheophilic fish

Resumo

O resfriamento do sêmen mantém espermatozóides viáveis ​​para estender o período útil para fertilização artificial e evita o uso de protocolos robustos e altos custos associados à criopreservação. No entanto, as curvas de qualidade espermática do sêmen fresco e refrigerado ainda não foram comparadas para o pacu (Piaractus mesopotamicus), que é frequentemente usado como modelo biológico. Este estudo teve como objetivo analisar a qualidade do sêmen de P. mesopotamicus durante 24 horas de refrigeração. Seis machos adultos foram induzidos com extrato de hipófise de carpa. Os dados de movimento espermático, integridade da membrana e morfologia foram comparados entre amostras de sêmen armazenadas por 24 horas em temperatura ambiente ou sob refrigeração a 12,63 °C. A motilidade dos espermatozoides diferiu significativamente ao longo do tempo. Após 24 horas de armazenamento, os valores de motilidade foram maiores nos espermatozoides refrigerados do que naqueles mantidos em temperatura ambiente. As taxas de sobrevivência dos espermatozoides não diferiram em 4 e 8 h após a coleta. Após 16 h, as células refrigeradas apresentaram integridade de membrana superior (82,05 ± 4,23%) em comparação àquelas armazenadas em temperatura ambiente (66,98 ± 6,45%), mantendo esse padrão até 24 h. Em termos de taxa de morfologia espermática, o sêmen de ambos os tratamentos ainda era viável para uso 8 horas após a coleta. Porém, após 16 h de armazenamento, ambos os grupos apresentaram grande redução nas taxas de normalidade, e às 24 h, todo sêmen estava inviável. Conclui-se que o sêmen de P. mesopotamicus pode ser armazenado até 8 h após a coleta quando refrigerado a 12,63 °C, sem uso de diluentes e/ou crioprotetores, mantendo qualidade suficiente para a fertilização de ovócitos.

Palavras-chave:
sêmen resfriado; análise espermática computadorizada assistida; reprodução de peixes; peixes reofílicos

1. Introduction

Refrigeration is frequently used to conserve fish milt over short periods of time (hours or days) to increase sperm viability yet avoid the need for cryopreservation, which is not only a more expensive technique but usually presents low conservation efficiency (Oliveira et al., 2007). Cooling fish milt is a simple technique that optimizes induced breeding in the laboratory and field by allowing the synchronization of male and female gamete availability at the time of spawning (Ninhaus Silveira et al., 2002; Horváth et al., 2003; Marques and Godinho, 2004; Linhart et al., 2005).

Cooling conserves milt by reducing the metabolic activity of spermatozoa at temperatures below physiological temperature (Sanches and Cerqueira, 2010), thereby extending the use of viable spermatozoa for hours in species such as piracanjuba (Brycon orbignyanus; Murgas et al., 2004) and pirapitinga (Brycon nattereri; Oliveira et al., 2007). Previous research has also assessed the effect of adding extenders at the time of refrigeration to improve the efficiency of sperm protection and thus ensure a longer viability period for fertilization, as was done for Colossoma macropomum (Pastrana, 2015), B. orbignyanus (Murgas et al., 2003), Mugil liza (Magnotti et al., 2019), and Lutjanus analis (Sanches and Cerqueira, 2011), among others.

Pacu (Piaractus mesopotamicus) is a neotropical teleost fish species belonging to the Characiformes family and is endemic to the La Plata River Basin (Flores Nava, 2007; FAO, 2010). Being widely distributed throughout South America, pacu is often used as a biological model. The species is rheophilic and, resultantly, has a defined reproductive period coinciding with environmental conditions that favor natural spawning and maximize larval survival. Spawning occurs between October and December, but it is more accentuated in November (Bernardino et al., 1988; Urbinati and Gonçalves, 2005). In a breeding environment, P. mesopotamicus is unable to perform reproductive migration, and females require hormonal induction to complete gamete maturation and reproduction. Although hormonal induction is a dominant process during breeding, some variables, such as the spawning synchronization of males and females, are difficult to control.

The application of cooling techniques for milt conservation provides one solution to the problem of spawning synchronization. P. mesopotamicus milt has been the subject of extensive explorative research, including an evaluation of different diluents for cooling (Streit Junior et al., 2007) and cryopreservation (Streit Junior et al., 2006; Teodozia et al., 2020), assessments of cryopreservation techniques (Paulino et al., 2012; Salmito Vanderley et al., 2012), and quality comparisons between fresh and cryopreserved sperm (Streit Junior et al., 2009). However, to date, no studies have compared the sperm quality curves of fresh and refrigerated milt of P. mesopotamicus, which is of paramount importance for producers who do not have access to robust cryopreservation protocols. In addition, different parameters of the milt refrigeration technique, such as storage temperature and cooling velocity, must be evaluated owing to their different particularities for each species. Thus, the aim of the present study was to evaluate the viability of P. mesopotamicus milt stored under refrigeration and at ambient temperature, without the use of extenders.

2. Material and Methods

2.1. Study site, sample population, and reproductive management

Milt was collected from adult P. mesopotamicus males (weighing 2.365 ± 0.44 kg; n = 6) at the Aquaculture Laboratory of São Paulo State University in Brazil. The methodology was approved by the Ethics Committee for the Use of Animals at the university (approval no. 01/2021). The experiment was carried in December in the middle of the reproductive period. The fish were placed in a single polyethylene box (750 L), weighed, and anesthetized with eugenol (50 mg/L). For hormone treatment, the fish received intraperitoneal injections of crude carp pituitary extract (EBHC) at a single dose of 5 mg/kg body weight, according to a protocol adapted from Araújo et al. (2014). After 240 degree-hours (at 27 °C), extrusion was performed through abdominal massage in the cephalocaudal direction.

2.2. Experimental design

Two milliliters of milt were collected from each of the six male specimens. Each sample was then separated into two 1 mL aliquots that were transferred to 1.5 mL microtubes. One microtube of milt from each male was stored under refrigeration in a thermal box containing ice (12.63 ± 2.55 °C), and the other microtube was stored at ambient temperature (25.23 ± 0.72 °C; Figure 1). Milt parameters were monitored at 4, 8, 16, and 24 h after collection, with the initial milt sample (0 h) serving as a control.

Figure 1
Experimental design for the sampling of Piaractus mesopotamicus milt, which was stored either at ambient temperature (25.23 ± 0.72 °C) or under refrigeration in a thermal box (12.63 ± 2.55 °C).

2.2.1. Sperm concentration

To measure the sperm concentration of a sample, 5 μL of milt was diluted in 5,000 μL of buffered saline formalin (1:1,000) at 4.6%. Subsequently, a Neubauer hematimetric chamber (0.1 mm deep) was used to conduct sperm cell counts, according to the methods of Sanches et al. (2011). The sperm concentration was then calculated as per the method used for mammalian semen recommended by the Brazilian College of Animal Reproduction (CBRA, 1998).

2.2.2. Sperm movement parameters

To activate sperm, 5 µL of milt and 100 µL of distilled water (a ratio of 1:20 for milt:water) were combined in a 1.5 mL microtube. An aliquot of this milt and distilled water solution was then deposited in a mirrored Neubauer chamber (0.1 mm deep) and covered by a glass coverslip (24 × 24 mm). The chamber was immediately observed under the 10× objective of a trinocular light microscope (Solaris, Bel Engineering, Monza, Italy) coupled with a Basler acA640-120gc camera (Basler, Ahrensburg, Germany), connected to the computer. Video record ing was initiated concomitant with the sperm activation using Pylon software (Basler, 2024) and extended up to 60 s (the time required for the loss of motility in all spermatozoa). All videos were processed as described by Wilson-Leedy and Ingermann (2007) using the Computer-Assisted Semen Analysis (CASA) plugin Wilson-Leedy and Ingermann (2006) in ImageJ open-source software (NIH, 2024). For this purpose, the configurations were adapted to those used for Brazilian neotropical fish (Sanches et al., 2010; Neumann et al., 2013). The analysis determined the number of mobile spermatozoa displaying values greater than 15, 10, and 3 μm/s for a curvilinear, average path, and straight-line velocity, respectively. Using the CASA plugin, the following variables could be determined: (1) the sperm motility rate (MOT, %), as the ratio between motile spermatozoa and the total number of spermatozoa in the field of view; (2) average path velocity (VAP, µm/s), calculated using the average displacement between individual points of the total sperm trajectory; (3) curvilinear velocity (VCL, µm/s), i.e., the cell velocity along its actual path of motion; (4) straight-line velocity (VSL, µm/s), i.e., the cell velocity considering the straight-line distance between the starting and end points of a trajectory; (5) cross-flagellar beat frequency (BCF, Hz), or the number of times the sperm head crossed the movement of direction; (6) straightness (STR, %), taken as the VSL/VAP ratio; (7) oscillation (WOB, %), comprising the oscillation between the actual trajectory and the average trajectory and calculated as the VAP/VCL ratio; and (8) progression (PROG) (Verstegen et al., 2002).

2.2.3. Membrane integrity rate

Sperm membrane integrity was analyzed using the protocol described by Bombardelli et al. (2006). In microtubes (1.5 mL), 25 μL of milt, 100 μL of 3% eosin blue, and 100 μL of 5% nigrosin were added. The contents of the tubes were homogenized and smears were prepared by adding a 10 μL aliquot of each sample to a slide. These smears were analyzed under a 40× light microscope, and for each slide approximately 600 cells were evaluated in three fields. The variable was expressed as percentage (%). Uncolored cells were considered to possess intact membranes, while red- or pink-stained cells were considered to be damaged (by exhibiting membrane permeability to the eosin-nigrosin dye).

2.2.4. Sperm morphology

To analyze sperm morphology, 10 μL of milt, 1,000 μL of buffered formalin with 4.6% saline solution, and 10 μL of Rose Bengal dye were added to microtubes (1.5 mL; Streit Junior et al., 2004). After homogenization, two 10 μL aliquots were placed parallel to one another on slides. The slides were then positioned “upright” at a 45° angle for 2-5 seconds. Two rows of samples were thus formed due to the effect of gravity on the aliquots (Sanches et al., 2016). The slide contents were analyzed under a light microscope with a 40× objective. Approximately 600 cells were counted in three different fields per slide. The variable was expressed as percentage (%). Sperm were classified as having a typical or atypical morphology following methodology adapted of Galo et al., 2009, considering as abnormal cells with broken, curled, degenerated, and bent tails, loose head and loose tail, micro and macrocephaly.

2.3. Statistical analysis

The data were subjected to assumptions of normality and homoscedasticity using Shapiro–Wilk and Levene tests. Outliers were determined by Cook's tests >0.1 and were removed. One-way analysis of variance (one-way ANOVA) was performed for all treatments, and the means were compared using Tukey's test (P < 0.01). Data were expressed as mean ± standard deviation. All analyses were performed utilizing the recommendations of Zar (2010) and using the Statistical Analysis System (Statistical Analysis System Institute, 2002-2003).

3. Results

3.1. Sperm concentration and movement parameters

The average sperm concentration observed in the present study was 3.71 ± 2.87 × 10⁹ spermatozoa/mL, this result is lower to that reported by Galo et al. (2019) of 21.09 × 10⁹ spermatozoa/mL. The difference in sperm concentration may be related to collections done in different reproductive stages of males. Regardless of the storage temperature, milt showed a decrease (P < 0.01) in motility rate compared to the control (0 h) at 4–24 h. However, when milt was stored at the lower temperature range (12.63 ± 2.55 °C), this observed decrease in motility rate occurred more slowly, from 91.73 ± 9.13% to 78.19 ± 11.73% up to 4 h, down to 65.17 ± 13.66% at 8 h, 33.86 ± 16.46% at 16 h, and 9.36 ± 5.68% at 24 h. In contrast, when stored at an ambient temperature range (25.23 ± 0.72 °C), the reduction in sperm motility rate was more accelerated, being 25.30 ± 18.99% after 4 h and practically zero (0.81 ± 1.70%) after 24 h (Figure 2).

Figure 2
Change over time for sperm activity parameters evaluated via Computer-Assisted Semen Analysis, using pacu milt either stored at ambient temperature (dark bars) or cooled in a thermal box (light bars): (A) sperm motility rate (%); (B) curvilinear velocity (VCL; µm s^-1); (C) average path velocity (VAP; µm s^-1); (D) straight-line velocity (VSL; µm s^-1); (E) oscillation (WOB; %); (F) straightness (STR; %); (G) progression (PROG, µm); and (H) cross-flagellar beat frequency (BCF, Hz). Average values followed by the same letter in the line do not differ based on results from conducting the Tukey’s test, when considering there were mean differences if there was a 0.01 P value.

VCL values were maintained in cooled milt for up to 8 h. In contrast, the VCL of milt kept at ambient temperature initially reduced rapidly up to approximately 4 h, reestablished itself after 8 h to attain values similar to those of the control and the refrigerated milt, but thereafter again decreased sharply. From 16 h onwards, refrigerated milt maintained higher VCL values (119.86 ± 28.67 μm/s at 16 h and 100.51 ± 22.19 μm/s at 24 h) than non-refrigerated milt (84.86 ± 8.79 μm/s at 16 h and a zero value at 24 h).

VAP was maintained for up to 4 h in the refrigerated milt (81.73 ± 13.06 μm/s) when compared to the control (93.49 ± 17.67 μm/s), but then reduced over time (66.32 ± 13.79 μm/s at 8 h, 56.57 ± 18.48 μm/s at 16 h, and 43.39 ± 15.37 μm/s at 24 h). In milt stored at ambient temperature, the VAP was lower (P < 0.01) than that of the control in the first 4 h of the experiment (54.92 ± 20.19 μm/s), showed an increase at 8 h after collection (62.97 ± 16.66 μm/s), and decreased again to 27.00 ± 11.49 μm/s at 16 h, reaching a zero value after 24 h. As with VCL, beyond 16 h of storage, refrigerated milt exhibited higher VAP values (61.49 ± 37.42 μm/s) than milt stored at ambient temperature.

Pacu milt maintained a constant VSL for up to 16 h when stored in a refrigerated environment. In contrast, the VSL value of milt decreased significantly between the 8 h (33.65 ± 10.89 μm/s) and 16 h (19.74 ± 13.25 μm/s) marks when it was stored at ambient temperature, reaching zero at 24 h. The refrigerated milt maintained a VSL value as high as 31.61 ± 12.39 μm/s, even after 24 h of storage.

WOB only showed a significant change after 16 h of storage. At this time point, the WOB of milt stored at ambient temperature was significantly less than that of similarly stored milt at 4 h post collection (Figure 2).

The spermatozoa of P. mesopotamicus moved in circular trajectories as the post-collection time passed (Figure 3). Coincidingly, the STR values were significantly lower in fresh milt from the control group (53.82 ± 9.40%) compared to that of refrigerated milt at 16 h (68.97 ± 19.65%). The STR of sperm kept at ambient temperature displayed the same behavior as that of refrigerated milt, except after 24 h, when the STR of milt stored at ambient temperature dropped to zero.

Figure 3
Sperm trajectories as recorded 10 s after sperm activation: (A) the control at 0 h; the sperm trajectories observed in milt stored at ambient temperature for (B) 4 h, (C), 8 h, (D) 16 h, and (E) 24 h; and sperm trajectories in milt after (F) 4 h; (G) 8 h; (H) 16 h; and (I) 24 h of refrigerated storage.

Overall, the PROG of stored milt decreased over time compared to that of the control (2,352.27 ± 621.53 µm). This decrease was only significant from 16 h onwards for refrigerated milt (1,661.86 ± 595.07 µm at 16 h and 1,450.98 ± 567.61 µm at 24 h), whereas it was already significant after 4 h for milt kept at ambient temperature (1,623.46 ± 473.94 µm at 4 h, 1599.37 ± 512.89 µm at 8 h, 929.26 ± 566.91 µm at 16 h, and zero at 24 h).

BCF values remained constant in the first 4 h post collection for refrigerated milt (44.04 ± 4.56 Hz, compared to the 37.52 ± 4.52 Hz of the fresh milt control); over the later time points, however, BCF increased (P < 0.01) in refrigerated milt, up to 52.79 ± 8.51 Hz at 24 h. Comparatively, the BCF of milt kept at ambient temperature increased (P < 0.01) in the first 4 h post collection (50.98 ± 7.42 Hz), then displayed no statistical difference with fresh milt until 16 h (49.82 ± 8.05 Hz), and ultimately dropped to zero at 24 h after collection.

3.2. Membrane integrity rate

No significant difference was observed between the membrane integrity of refrigerated milt and that of the control during the entire 24 h observation period. In contrast, the membrane integrity of milt kept at ambient temperature decreased (P < 0.01) at 16 h (66.98 ± 6.45%) compared to that of the control (86.41 ± 4.60%), but then remained stable (P > 0.01) up to 24 h post collection (67.66 ± 7.64%), as illustrated in Figure 4.

Figure 4
Change over time in (A) the sperm morphology or normality sperm rate (%) and (B) the membrane integrity or survival rate (%) of pacu milt stored at either ambient temperature (dark bars) or under refrigeration in a thermal box (light bars). Average values followed by the same letter in the line do not differ based on results from conducting the Tukey’s test, when considering there were mean differences if there was a 0.01 P value.

3.3. Sperm morphology

No statistical difference was observed between the sperm morphology rates of the two treatment groups. However, when comparing the sperm morphology rates of the two treatment groups with that of the control, the normality rate clearly remained stable (P > 0.01) for up to 8 h in milt maintained at ambient temperature (22.75 ± 8.15%) and up to 16 h in refrigerated milt (33.72 ± 4.76%). The normality rate of refrigerated milt only decreased significantly after 24 h of storage, while that of milt kept at ambient temperature already decreased (P < 0.01) after 16 h (22.75 ± 8.15%), as depicted in Figure 4).

The main spermatozoan abnormalities observed after 8 h of storage were tail defects, mainly comprising short or bent tails (Figure 5). After 16 h, the presence of spermatozoa with loose heads increased, in addition to the aforementioned tail defects, with a large number of isolated tails and heads being observed.

Figure 5
Image of a slide representing some of the most common morphological abnormalities found in pacu milt samples: (A) the presence of gout in the proximal tail region; (B) spermatozoon with a short tail; (C) a tail bent in the proximal region; (D) a tail bent in the middle region; and (E) a tail bent in the distal region. Dye: Rose Bengal. Magnification: 40×.

4. Discussion

Monitoring seminal viability is important for the fish production sector as it provides key information such as the maximum milt storage time. A lack of synchrony between spawning and spermiation can cause damage at the time of oocyte fertilization; applying techniques that preserve the viability of male gametes for a longer period can help overcome this obstacle.

Good results after storing P. mesopotamicus milt for 30 h in a domestic refrigerator were reported (Lima et al., 1989); however, motility values were not reported. In the present study, we observed that pacu milt storage at ambient temperature negatively affected sperm motility after 4 h, with the mean motility values obtained for fresh milt being consistent with previously reported values of 98.7% (Teodozia et al., 2020), 95.3% (Silva et al., 2022), and 95.0% (Maria et al., 2004) using the same methodology. This observation is of great importance, considering that sperm motility plays a crucial role in fertilization success (Bobe and Labbé, 2010). Milt samples kept at ambient temperature were unfeasible (<20%) at all times, while refrigerated samples showed motility rates in excess of 60% at 8 h post collection. This sperm motility value for refrigerated milt can be considered satisfactory, since it is assigned as a limit for milt utilization, with a sperm motility rate of at least 30% being required for different species (Marques and Godinho, 2004). Similar results were observed for sperm motility rates of pacu milt kept under refrigeration for up to 19 h, during which there was a constant reduction in this parameter over time (Marques and Godinho, 2004). Pacu milt was evaluated cooling over 120 h using different extenders and samples with 30% motility after up to 72 h of storage was obtained (Streit Junior et al., 2006).

In other species, tambaqui (C. macropomum) milt has been demonstrated to display 0% motility after 24 h of storage under refrigeration at 4 °C (Pastrana, 2015). In the present study, a motility of 9.36 ± 5.68% was observed after 24 h of storage, which is below the rate of 23.75% reported by Santana (1998) after storing pacu milt for 24 h at 4 °C. These divergent results indicate that there is a great variation in sperm motility between fish species. Some studies have only verified motility over a short observational period. For example, sperm motility in the refrigerated milt of carp (Cyprinus carpio) was verified only for up to 11 h of storage (Padua et al., 2012). However, for other species, such as piracanjuba (Brycon orbignyanus), sperm motility has been observed for up to 144 h under refrigeration using BTS as a diluent (Murgas et al., 2004). The differences recorded for pacu milt in the present study in relation to those found in the literature may be associated with (i) the methodology applied for temperature reduction, (ii) species-specific differences in milt characteristics, or (iii) the protocol and dosage used.

Although there was a small reduction in the sperm motility of refrigerated milt after 4 and 8 h of storage compared to that of freshly collected milt, milt refrigeration was nevertheless effective, considering that sperm motility dropped drastically (<20%) in milt kept at ambient temperature for 4 h. Interestingly, the motility of milt cooled for 4–8 h is adequate to maintain the fertilization rate of the tetraodontid Takifugu niphobles (Gallego et al., 2013). The indication that sperm motility in pacu milt was adequate for up to 8 h of refrigerated milt storage in this study (although with a small reduction in relation to motility at time zero) was corroborated by the maintenance of other parameters obtained via CASA, such as the maintenance of VCL (for up to 8 h), VSL (up to 16 h), WOB (up to 24 h), and PROG (up to 8 h).

Although the VAP characteristic was maintained for up to 4 h in refrigerated pacu milt, the corresponding reduction in VAP observed at other time points was smaller than that observed in milt kept at ambient temperature; in this regard, as well as for the VSL, WOB, STR, PROG, and BCF characteristics, only the refrigerated milt showed values for these variables, corroborating that milt refrigeration provided partial maintenance of qualitative characteristics at 16 and 24 h. Notably, values of the STR variables increased at 16 h, and only those of the refrigerated milt remained high (they were zero in milt kept at ambient temperature). Furthermore, the BCF increased in milt maintained at ambient temperature for 4 and 16 h of storage, with a zero value at 24 h, and also increased in milt kept refrigerated for 8 h, maintaining this increase up to 24 h. Appropriate transposed beat frequencies, oscillation, progression and straightness are associated with more efficient movements, allowing sperm to swim faster and in a straighter line, which increases their chances of successful fertilization, high values ​​indicate that the sperm is consuming energy, which is a reflection of good cellular health and adequate mitochondrial function, however very high values, in excess, may indicate that there is excessive energetic activity that can reduce the longevity of sperm (Amann and Katz, 2004; Mortimer, 1997; Aitken and Baker, 2006).

Considering that fertilization during artificial breeding is performed dry (via the addition of milt to oocytes without the presence of water), rates of sperm membrane integrity and sperm normality may have a greater influence on overall seminal viability, even more so than indices such as motility rate. We observed low motility values valid for non-refrigerated milt at 16 h after collection; however, positive results were recorded for the survival rates and sperm normality at this time point. Thus, this milt could be considered viable. After 24 h of refrigeration, we detected a large number of decapitated spermatozoa; such a characterization may exert a negative effect at the time of fertilization, in addition to the low motility observed at this point (9.36 ± 5.68%).

Membrane integrity rates are related to cellular membrane damage, which influences the survival rate negatively. These values decrease over time, and a small percentage of spermatozoa with defects can nevertheless fertilize oocytes, promoting a drop in the hatching rate and causing problems in the initial development of eggs (Pérez Cerezales et al., 2010). In this study, spermatozoan abnormalities appeared after 8 h of storage and initially consisted of tail defects, mainly short or bent tails, and expanded at 16 h to include decapitated spermatozoa.

5. Conclusion

P. mesopotamicus milt can be stored successfully up to 8 hours after collection when refrigerated at 12.63 °C, without the need for extenders and/or cryoprotectants. Under these conditions no spermatozoa membrane integrity change occurred, and enough motility and spermatozoa normal morphology was maintained for possibles favorable egg fertilization.

Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES), Finance Code 001, Federal University of Mato Grosso do Sul (UFMS).

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Publication Dates

• Publication in this collection
  21 Oct 2024
• Date of issue
  2024

History

• Received
  05 June 2024
• Accepted
  15 July 2024