Di Federico F.1, Mattioli S.1, Angelucci E.1, Agradi S.2, Fehri N.E.2, Castellini C.1, Dal Bosco A.1
- Department of Agricultural, Environmental and Food Science, University of Perugia, Italy.
- Department of Veterinary Medicine and Animal Science, University of Milano.
ABSTRACT
Forty-five New Zealand White rabbit bucks were used for the experimental trial (15 rabbit/groups). Three different diets were administered ad libitum for 110 days: standard diet (C), the same diet with 5% of linseed (L), and another one with 5% linseed + 0.2% of Padina pavonica algae extract (LPP). The semen was collected twice per week and volume, concentration, motility, vitality, capacitation and acrosome reaction were evaluated. The oxidative status and the fatty acids were also assessed by spectrophotometer and chromatography analysis, respectively. The aim of research is to test the use of an algae (Padina pavonica) extract as an enhancer of long-chain fatty acids-metabolism in combination with a limited amount of linseed. The results showed that the use of flaxseed (5%) combined with 0.2% algae extract (LPP) did not have a significant effect on the main kinetic traits and fatty acid profile of sperm, compared to L group, whereas, after 8 wk. of administration, there was an increase in the quantity of n-3 PUFA compared to the control group.
Keywords: Rabbits, Polyunsatured Fatty Acids, Padina Pavonica, Lineseed, Reproduction, Meat Quality.
INTRODUCTION
Linseed supplementation is considered a useful strategy for improving the reproductive parameters of male and female rabbits. Literature reported that about 10% dietary supplementation of linseed administered for 110 days modifies the fatty acid profile and the semen quality of rabbit bucks (Castellini et al., 2021). However, this level of inclusion is not always economically sustainable, thus also other dietary strategies should be proposed. In this context, the purpose of this research is to test the use of an algae (Padina pavonica) extract as an enhancer of long-chain fatty acids metabolism in combination with a little amount of linseed (5%) with the aim to improve the semen characteristics of rabbits without increase a lot the cost of feed.
MATERIALS AND METHODS
Animals and experimental design
At the Department of Agricultural, Environmental and Food Science of Perugia University rabbitry, forty-five New Zealand White rabbit bucks (6 months of age) were divided into 3 experimental groups (Table 1): Control group (C) fed a standard diet, Linseed group (L) fed a standard diet enriched with 5% of linseed and the flax-algae group (LPP) fed standard diet plus Padina Pavonica algae at 0.2% and linseed at 5%. The diets were formulated to be isoenergetic, and isoproteic (Martini s.r.l) and were administered for a total of 120 days (60d of adaptation + 60d experiment). Semen collection was performed twice every week.
Table1: Formulation and composition ( %, UI, Kcal) of diets
| C | L | LPP | C | L | LPP | |||||
| Ingredients | ||||||||||
| Wheat bran | 25.1 | 24.8 | 24.9 | C. Protein | 16.5 | 16.5 | 16.5 | |||
| Barley | 13.3 | 13.0 | 13.0 | Lipids | 3.62 | 3.93 | 3.93 | |||
| Sunflower seed | 12.0 | 11.7 | 11.5 | Fiber | 17.16 | 16.79 | 16.79 | |||
| Alfalfa meal | 10.8 | 13.0 | 13.0 | Ash | 7.99 | 8.09 | 8.09 | |||
| Sunflower husks | 10.0 | 10.0 | 10.0 | DE (kcal) | 2350 | 2350 | 2350 | |||
| Beet pulp | 7.5 | 5.7 | 5.5 | |||||||
| Extruded linseed | – | 5.0 | 5.0 | |||||||
| Full-fat soybean | 5.0 | 2.9 | 3.1 | |||||||
| Wheat straw | 4.2 | 2.0 | 2.0 | |||||||
| Molasses cane | 3.0 | 3.0 | 3.0 | |||||||
| Wheat | 2.5 | 2.5 | 2.5 | |||||||
| Grape seed meal | 2.3 | 1.7 | 1.7 | |||||||
| Soya hulls | 0 | 1.7 | 1.7 | |||||||
| Calcium carbonate | 1.6 | 1.5 | 1.4 | |||||||
| Soybean oil | 0.78 | – | – | |||||||
| Sodium chloride | 0.40 | 0.40 | 0.40 | |||||||
| Palm oil | 0.33 | – | – | |||||||
| Carboxymethylcellulose | 0.30 | 0.30 | 0.30 | |||||||
| Mineral-vitamin premix1 | 0.25 | 0.25 | 0.25 | |||||||
| Alga PP | – | – | 0.20 | |||||||
| Lysine HCl | 0.16 | 0.17 | 0.17 | |||||||
| Liquid acidifier2 | 0.15 | 0.15 | 0.15 | |||||||
| Magnesium oxide | 0.1 | 0.1 | 0.1 | |||||||
| Methionin | 0.06 | 0.07 | 0.07 | |||||||
| Choline | 0.05 | 0.05 | 0.05 | |||||||
| Vitamin E 50% | – | 0.03 | 0.03 | |||||||
| L Threonine | 0.03 | 0.01 | 0.01 | |||||||
| DL Methionine | 0.03 | – | – | |||||||
Semen sampling and qualitative characteristics evaluation
The semen was collected with an artificial vagina in different glass tubes for each rabbit and then stored in eppendorf. The samples were immediately transported to the laboratory of the University of Perugia where qualitative analyses were performed. The sperm kinetic traits were evaluated with CASA System (ISAS©, Valencia, Spain).
Chemical Analyses
The oxidative status and fatty acid profile of rabbit semen were assessed by spectrophotometer (Ke et al, 1997) and chromatography (Folch et al, 1961) analysis respectively, while the tocols profile of semen and blood plasma (tocols) was assessed by HPLC (Hewavitharana et al, 2004) at 0, 4 and 8 weeks of dietary administration.
Statistical Analysis
The statistical analysis was done with a mixed linear model considering the repeated effect of buck and the effects of the time of collection, diet and their interaction. The post-hoc Bonferroni test were used for defining the significance of the differences (p<0.05).
RESULTS AND DISCUSSION
Table 2 shows the main traits of rabbit semen. All the effects have been reported (diet, time and their interaction), however, only the effect of diet will be discussed in detail. There are no differences between experimental groups regarding volume, live and dead cells, static sperm, non progressive motility, curvilinear velocity (VCL), oscillation index (WOB) and the movements of the sperm head (ALH).
Semen concentration and progressive motility were higher in L and LPP groups as well as other kinetic parameters (linearity-LIN, straight trajectory-STR, and flagellum beat cross frequency -BCF). The L group also increases the BCF which could indicate a pre-capacitative status of sperm cells.
The reasons for this light improvement can be traced back to a probable modofied asset of PUFA profile which affects the lipid structure of the membranes (Mourvaki et al. 2010) and at the same time improves their elasticity and motility.
Table 2. Semen quality evaluations of rabbit fed experimental diets
| Parameters | Unit | C | L | LPP | RMSE | P | ||
| Diet | Time | DxT | ||||||
| Volume | ml | 0.30 | 0.30 | 0.38 | 0.13 | 0.115 | 0.018 | 0.249 |
| Concentration | x 10^6/ml | 509.36a | 576.05b | 595.08b | 3.93 | 0.046 | 0.385 | 0.408 |
| Live cells | % | 78.21 | 80.00 | 78.70 | 1.06 | 0.686 | <0.001 | 0.318 |
| Dead cells | “ | 21.78 | 20.00 | 21.29 | 1.06 | 0.686 | <0.001 | 0.318 |
| Static | “ | 25.00b | 18.85a | 20.00a | 0.11 | 0.087 | 0.002 | 0.004 |
| Motile non progressive | “ | 64.00 | 60.00 | 63.00 | 0.11 | 0.384 | 0.007 | <0.001 |
| Motile progressive | “ | 11.00a | 20.00b | 16.50ab | 0.1 | 0.001 | 0.008 | <0.001 |
| VCL | mm/s | 239.10 | 242.16 | 230.98 | 1.93 | 0.724 | <0.001 | 0.035 |
| LIN | % | 19.83a | 24.8b | 21.81ab | 0.67 | <0.001 | 0.011 | 0.003 |
| STR | % | 42.38a | 52.27b | 44.77a | 0.95 | 0.01 | 0.01 | <0.001 |
| WOB | % | 47.70 | 47.39 | 49.06 | 0.64 | 0.118 | <0.001 | 0.058 |
| ALH | mm | 3.22 | 3.47 | 3.12 | 0.24 | 0.013 | 0.714 | 0.245 |
| BCF | Hz | 16.39a | 25.34b | 18.52ab | 0.85 | <0.001 | <0.001 | <0.001 |
On the same row different letters (a, b) means P<0.05 for the effect of diet
The fatty acid profile of semen (Table 3) was not affected by DietXTime interaction.
Instead, we had a significant difference related to the dietary effect on quantitative of docosahexaenoic acid (DHA-C22:6n-3) which is higher in the LPP group after 8 weeks of administration, than others. While the time effect affects the quantitative of Polyunsaturated fatty acids (PUFA) and PUFA n-3, always from 8 weeks of administration.
Table 3. Oxidative status (TBARS, nmol MDA/mL) and fatty acid profile (% total fatty acids) of rabbit semen at 0, 4 and 8 weeks of dietary administration.
| 0 | 4 wk | 8 wk | RMSE | P | |||||||||
| C | L | LPP | C | L | LPP | C | L | LPP | Diet | Time | DxT | ||
| TBARS | 4.46 | 14.15 | 13.16 | 4.07 | 12.43 | 12.36 | 8.74 | 12.06 | 17.40 | 1.00 | 0.969 | 0.820 | 0.388 |
| C18:2n-6 | 8.06 | 7.69 | 4.96 | 7.91 | 7.02 | 7.89 | 7.58 | 7.51 | 8.76 | 0.59 | 0.245 | 0.650 | 0.125 |
| C18:3n-3 | 0.15 | 0.34 | 0.18 | 0.00 | 0.26 | 0.25 | 0.00 | 0.17 | 0.28 | 0.82 | 0.136 | 0.162 | 0.131 |
| C20:3n-3 | 1.07 | 0.77 | 0.79 | 0.66 | 0.78 | 0.33 | 0.64 | 0.99 | 0.99 | 0.27 | 0.255 | 0.482 | 0.319 |
| C20:4n-6 | 1.29 | 0.96 | 0.96 | 1.08 | 0.84 | 0.49 | 1.10 | 1.28 | 1.20 | 0.20 | 0.654 | 0.720 | 0.356 |
| C20:5n-3 | 1.07 | 0.88 | 0.95 | 0.83 | 0.66 | 0.64 | 0.23 | 0.50 | 0.23 | 0.59 | 0.181 | 0.259 | 0.079 |
| C22:5n-6 | 19.66 | 17.7 | 17.84 | 16.04 | 16.95 | 11.84 | 23.75 | 23.36 | 23.55 | 0.92 | 0.182 | 0.593 | 0.399 |
| C22:5n-3 | 2.22 | 1.51 | 1.57 | 1.82 | 1.76 | 1.20 | 2.07 | 2.68 | 2.25 | 0.56 | 0.697 | 0.610 | 0.544 |
| C22:6n-3 | 0.65c | 0.51c | 0.79c | 0.80c | 0.62c | 0.31b | 0.10a | 0.18a | 0.39b | 0.18 | 0.004 | 0.243 | 0.118 |
| SFA | 46.7 | 47.71 | 50.68 | 47.12 | 51.88 | 52.23 | 49.1 | 48.63 | 46.42 | 0.26 | 0.275 | 0.278 | 0.987 |
| MUFA | 3.85 | 8.02 | 8.79 | 7.73 | 7.22 | 16.63 | 5.86 | 5.55 | 8.12 | 0.25 | 0.407 | 0.468 | 0,228 |
| PUFA | 34.17 | 30.36 | 28.04 | 29.14 | 28.89 | 22.95 | 35.47 | 36.67 | 37.66 | 0.27 | 0.906 | 0.002 | 0.390 |
| PUFAn-3 | 2.94 | 2.50 | 2.71 | 2.29 | 2.32 | 1.53 | 0.97 | 1.84 | 1.89 | 0.97 | 0.668 | 0.043 | 0.220 |
| PUFAn-6 | 31.23 | 27.86 | 25.33 | 26.85 | 26.57 | 21.42 | 34.50 | 34.83 | 35.76 | 0.36 | 0.345 | 0.189 | 0.394 |
on the same row different letters (a,..c) means P<0.05 for Diet
The tocol profile (Table 4) showed a different trend in semen and blood. In semen the highest value was found in L groups than C and LPP after 4 wk., with a subsequent reduction at 8 wk., whereas in blood the highest value was found in LPP one, followed by L and C.
Table 4. Total Tocol content (nmol/mL) of rabbit semen and blood after 0, 4 and 8 weeks of administration.
| wk 0 | wk 4 | wk 8 | RMSE | P | |||||||||
| C | L | LPP | C | L | LPP | C | L | LPP | Diet | Time | DxT | ||
| Tocols semen | 1.01a | 1.39a | 1.39a | 1.22a | 1.89b | 1.38a | 1.69b | 0.88a | 0.83a | 0.356 | 0.025 | 0.056 | <.001 |
| Tocols blood | 0.72b | 0.74b | 0.80b | 0.40a | 1.18b | 2.60c | 0.84b | 0.30a | 0.77b | 0.14 | <.001 | <.001 | <.001 |
on the same row different letters (a, b) means P<0.05 for Diet
CONCLUSIONS
In this study, the effect of two sources of PUFA (linseed combined or not with algae extract) in feeding of rabbit bucks were studied. The results showed that the use of low amount of linseed (5%) combined or not with 0.2 % of algae extract (due to the economic sustainability of the supplementation) did not improve the most relevant semen traits.
With respect to other studies, which used higher amount of linseed, nor the kinetic traits nor the fatty acid profile of semen were greatly affected by treatment. Accordingly, the expected enhancing effect of algae on rabbit buck metabolism was not recorded. Probably, higher amount of linseed and algae extract are required, or a prolonged time of administration.
Further studies to deepen the algae linseed interaction on rabbit metabolic response (blood, organs) are needed.
AKNOWLEDGEMENT
Research was funded by PRIMA ΩRABBIT project.
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